universidade federal do cearÁ centro de ciÊncias …À professora selma mazzetto por sempre manter...
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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
FORTALEZA-CE
2016
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
FORTALEZA-CE
2016
Ao meu amigo Afrânio, autor da
inesquecível frase “daqui a dez anos
seremos todos doutores”.
Exatamente dez anos se passaram
desde então!
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.
À inesquecível turma de Química Inorgânica Avançada: Samuel Victor,
Samuel Santos, Walysson, Renato e Felipe, onde passamos tantas manhãs e
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.
Aos ex-funcionários do departamento de físico-química, Wanda e
Ivanílson. Não houve um dia sequer que eu não os visse sempre de bom humor
e esbanjando humildade e cortesia. O trabalho desempenhado por ambos fora -
sem dúvidas - sem tamanho e de imensa importância para o funcionamento de
nosso laboratório. O departamento que hoje os tiver como funcionários é um
departamento de sorte.
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!
À turma do “racha da química”, pois mesmo em momentos de
descontração e lazer, surgiam discussões relevantes acerca de nossas teses,
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 minha banca de exame de qualificação, composta pelos
professores Dr. Audísio Filho, Dr. Amauri Jardim e Drª Judith Feitosa, que muito
contribuíram para o aperfeiçoamento e correção deste trabalho.
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!
Muito obrigado a todos!!
“Demore o tempo que for para decidir
o que você quer da vida, e depois que
decidir não recue ante nenhum
pretexto, porque o mundo tentará te
dissuadir.” (Friedrich Nietzsche)
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.
Palavras-chave: nanopartículas de prata; síntese verde; açúcares redutores.
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.
Keywords: silver nanoparticles; green synthesis; reducing sugars.
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 AgXC-01, AgXC-02, AgXC-03, AgXC-
04, AgXC-05 and AgXC-06.
59
Figure 2.7 – UV-Vis specter of samples without sodium citrate. 60
Figure 2.8 – UV-Vis specter of sample with sodium citrate only. 61
Figure 2.9 – FWHM of an arbitrary measure. 62
Figure 2.10 – XRD pattern for AgRC-01, AgRC-02 and AgRC-03. 64
Figure 2.11 – Size distribution by intensity of light scattering. 65
Figure 2.12 – Zeta potential of AgXC-05 sample. 67
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 4.5 – XRD. 106
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 6.3 – XRD of samples (a) centrifuged AgNPs, (b) AgNPs +
Itraconazole 3.2 µg mL-1 and (c) AgNPs + Itraconazole 4.8 µg mL-1.
147
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
Figure 6.11 – Susceptibility testing antifungal agents. 157
Figure 6.12 – AgNPs + ITR representative scheme. 158
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
stability testing AgNPs content (%)a
129
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
3.4 Scanning electron microscopy (SEM) 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
1 INTRODUCTION 118
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
REFERENCES 159
CHAPTER 1
NANOTECHNOLOGY, SILVERNANOPARTICLES AND
GREEN SYNTHESIS: AN OVERVIEW
20
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 = 10-9m) 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.
Source: adapted from Castro [3]
21
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
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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.
There is a large variety of synthesis routes to obtain nanostructured
materials, as well as several applications. Large had been the development of
nanorobots [11], nanostructured devices that have a specific functionality. By
using nanorobots techniques, the medicine professionals are equipped with a
powerful tool that can make all the difference on treatment of diseases and
disorders in living organisms. These nanorobots may have the action defined by
target cells, organs or may be specifically directed to a region of organism trough
markers [12-15]. This direction may happen also by mechanical ways, such as
magnetic field, for example [16-20]. An area that reveals a great interest is
healthiness. The employment of nanotechnology has gained prominence along
the years, in a way that today there is a specific area: nanomedicine [8, 21-25].
23
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 shows various silver nanoparticles solutions with different
aspects of color. The solutions of different colors are derived from the different
morphology of the nanoparticles, as well as their different distributions of particle
size.
24
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:
a) Method of polysaccharides: the silver nanoparticles are prepared
through the use of polysaccharides as stabilizing agents and, in some
25
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].
d) Biological method: this is the use of living organisms extracts. Such
extracts can act both as reducing agent and as a stabilizing agent in
the synthesis of nanoparticles. The reduction of silver ions occurs
through the combination of biomolecules found in the reaction
medium, such as enzymes, proteins, amino acids, polysaccharides
and vitamins [40, 41]. Such syntheses are chemically benign,
although quite complex.
26
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.
The sugars are water soluble carbohydrates. One can divide these
carbohydrates to monosaccharaides, oligosaccharides and polysaccharides. The
first are the basis of all carbohydrates, forming units and these are also called
simple sugars. Among simple sugars, the smaller has only three carbons and
trioses are called, for example, glyceraldehyde, illustrated in Figure 1.4a.
Oligosaccharides are carbohydrates formed by some (few) linked
monosaccharaides, while polysaccharides are formed by several
monosaccharaides linked. These last are the most common carbohydrates such
as glycogen, starch, and cellulose, shown at Figures 1.4b, 1.4c and 1.4d
respectively.
27
Figure 1.4 – Chemical structure of sugars monomers: gliceraldehyde (a),
glycogen (b), starch (c) and cellulose (d).
HOH
OH
O
H
(a)
O
OH
OH
OHOH
(b)
OH
O
H
H
OH
OHOH
(c)
O
OH OH
OH
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.
Three sugars are particularly of interest in this work: ribose, arabinose
and xylose. They are all pentose and epimers each other. Their structures are
shown in Figure 1.5.
28
Figure 1.5 – L-arabinose (a), D-ribose (b) and D-xylose (c).
OH
OH
OH
H
H
H
OH
O H
(a)
OH
OH
H
H
H
OH
OH
O H
(b)
OH
H
H
OH
H
OH
OH
O H
(c)
Source: author
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:
Ag+(aq) + e- Ag(s) (Eo
red = +0,80V) (1.1)
29
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.
As the object of study of this work, it is included microorganisms from the
different kingdoms of monera (bacteria) and fungi (fungi). Three bacteria
belonging to the study scope of this study: Escherichia coli, Pseudomonas
aeruginosa and Staphylococcus aureus. For the kingdom fungi, Candida
albicans, Candida tropicalis, Candida parapsilosis, Aspergillus niger, Aspergillus
fumigatus and Aspergillus terreus.
30
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
There were found 30,307 published works among 2000 and 2015, which
gives an average of 1,894 works by year, which reveals an intense search for
knowledge of the most diverse properties and applications, in special in the last
five years.
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4000
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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].
The silver salts activity against bacteria has been reported since antiquity
[61] and silver is currently used to control bacterial growth in a variety of
applications, including dental work, catheters and also burns [62, 63]. Silver ions
and compounds containing silver are actually extremely toxic to microorganisms
showing various biocides in bacteria [64]. In a study by Aymonier and coworkers,
AgNPs functionalized amphiphilic macromolecules were associated with
antimicrobials as capping agents [65]. Silver nanoparticles have a more
pronounced activity against microorganisms when compared to its ions and silver
salts [66-69]. In addition to having activity against bacteria and viruses [64, 68,
70, 71] it has also been used to treat immunological and inflammatory disorders
[72]. The same nanoparticles find use in growth control mosquito vectors of
malaria, filariasis and dengue [73]. For these reasons, research on metal
nanoparticles, especially silver, has increased substantially in recent years [74,
75]. Other metal nanoparticles (which have a high surface area and a reasonable
amount of surface atoms) enter the list of research because of their unique
physicochemical properties such as catalytic activity, optical and electronic
properties, antimicrobial activity and magnetic properties [76-78]. Among the
metal nanoparticles, the silver are those with the best bacteriostatic and
bactericidal activity [79]. It is expected that the high surface area and their high
32
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].
The chemical synthesis of silver nanoparticles depends on a reducing
agent to give electrons and reduce silver ions, as already shown at equation 1.1.
Several routes to obtain silver nanoparticles are used, and some of them will be
described in this work.
33
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
When formed, nanoparticles are thermodynamically unstable and there
is a natural tendency to the agglomeration of particles and their consequent
growth, which increase the diameter, causing the phenomenon of polydispersity
and decant the solution. The great defy is to develop a way to avoid these
adversities and obtain a stable solution, as well as monodispersive system and
linear properties [107, 108].
34
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].
The steric stabilization may be performed by polymers, such as PVP
(polyvinylpyrrolidone), surfactants, such as sodium dodecyl sulfate and ligands,
which are molecules containing amine groups, phosphine and thiols. Figure 1.9
shows different types of stabilizations [111, 112].
35
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
OHO
-
O
O
O-
Na+
Na+
Na+
(a)
CH3
OS
O
O
O-
10
Na+
(b)
Source: author
To better understand the importance of capping agents, a scheme is
drawn at Figure 1.11, which shows the citrate ion capping AgNP (a) to avoid the
agglomeration (b) and consequent precipitation.
36
37
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])
Yang and co-workers [114] studied the effects of different stabilizers
agents over silver nanoparticles synthesis. In their work, the researchers used
PVP, sodium dodecyl sulfate and Arabic gum. Lah and co-workers [115] obtained
silver nanoparticles using Daxad 19 surfactant to control its shape and properties.
Hebeish [116] used starch to synthesize and cap silver nanoparticles, obtaining
spherical particles with size about 80nm and with a zeta potential about -25mV.
Vanamudan and co-workers synthesized spherical AgNP trough the utilization of
guar gum as capping agent [117].
38
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
Mie showed on his theory that the shape of particle, dielectric constant of
medium, such as temperature and refraction index of solvent also plays a role on
band displacement. His theory was developed starting from Maxwell’s laws and
shows agreement with experimental data [119].
39
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:
- Antimicrobial agent (against fungi and bacteria);
- Effect of addition (with antifungal Itraconazole).
40
REFERENCES
1. Feynman, R.P., There's plenty of room at the bottom. J MEMS, 1992. 1:
p. 60-66.
2. Taniguchi, N., Bulletin of the Japan Society of Precision Engineering,
1974: p. 18-23.
3. Castro, F.M.B.G.R., Nanotecnología, hacia un nuevo portal científico-
tecnológico. QuímicaViva, 2012. 3(11): p. 171-184.
4. Brasil. Relatório referente à gestão do programa "Desenvolvimento da
Nanociência e da Nanotecnologia" no exercício de 2005. 2008 [cited 2015
9 de maio].
5. Brasil. Site Portal Brasil. 2015 [cited 2015 9 de maio].
6. Brasil. Investimento em nanotecnologia pode chegar à R$ 110 milhões.
2012 [cited 2015 9 de maio].
7. Gharagozloo, M., S. Majewski, and M. Foldvari, Therapeutic applications
of nanomedicine in autoimmune diseases: From immunosuppression to
tolerance induction. Nanomedicine: Nanotechnology, Biology and
Medicine, 2015. 11(4): p. 1003-1018.
8. Gupta, A.S., Nanomedicine approaches in vascular disease: a review.
Nanomedicine: Nanotechnology, Biology and Medicine, 2011. 7(6): p.
763-779.
9. Bell, I.R., Advances in integrative nanomedicine for improving infectious
disease treatment in public health. European Journal of Integrative
Medicine, 2013. 5(2): p. 126-140.
10. Zhu, X., Nanomedicine in the management of microbial infection –
Overview and perspectives. Nano Today, 2014. 9(4): p. 478-498.
11. Jr, R.A.F., What is nanomedicine? Nanomedicine, 2005. 1(1): p. 8.
12. Chen, Y.T., Multiplexed quantification of 63 proteins in human urine by
multiple reaction monitoring-based mass spectrometry for discovery of
41
potential bladder cancer biomarkers. Journal of Proteomics, 2012. 75(12):
p. 3529-3545.
13. Drake, P.M., A lectin affinity workflow targeting glycosite-specific, cancer-
related carbohydrate structures in trypsin-digested human plasma.
Analytical Biochemistry, 2011. 408(1): p. 71-85.
14. Hang Xing, K.H., Ji Li, Seyed-Fakhreddin Torabi, Yi Lu, DNA Aptamer
Technology for Personalized Medicine. Curr Opin Chem Eng, 2014. 1: p.
9.
15. Misra, R.V., Developing an integrated proteo-genomic approach for the
characterisation of biomarkers for the identification of Bacillus anthracis.
Journal of Microbiological Methods, 2012. 88(2): p. 237-247.
16. Antônio C. H. Barreto, V.R.S., Rafael M. Freire, Selma E. Mazzetto,
Juliano C. Denardin, Giuseppe Mele, Igor M. Cavalcante, Maria E. N. P.
Ribeiro, Nágila M. P. S. Ricardo, Tamara Gonçalves, Luigi Carbone,
Telma L. G. Lemos, Otília D. L. Pessoa, Pierre B. A. Fechine Magnetic
Nanosystem for Cancer Therapy Using Oncocalyxone A, an Antitomour
Secondary Metabolite Isolated from a Brazilian Plant. Int. J. Mol. Sci.,
2013. 14(9): p. 15.
17. Murase, K., Control of the temperature rise in magnetic hyperthermia with
use of an external static magnetic field. Physica Medica: European Journal
of Medical Physics. 29(6): p. 624-630.
18. Vanessa Zamora-Mora, M.F.-G., Julio San Romána, Gerardo Goya,
Rebeca Hernández, Carmen Mijangos, Magnetic core–shell chitosan
nanoparticles: Rheological characterization and hyperthermia application.
Carbohydrate Polymers, 2014. 102: p. 8.
19. Lai, J.J., Multifunctional magnetic plasmonic nanoparticles for applications
of magnetic/photo-thermal hyperthermia and surface enhanced Raman
spectroscopy. Journal of Magnetism and Magnetic Materials, 2013. 331:
p. 204-207.
42
20. Manh, D.H., Structural and magnetic study of La0.7Sr0.3MnO3
nanoparticles and AC magnetic heating characteristics for hyperthermia
applications. Physica B: Condensed Matter, 2014. 444: p. 94-102.
21. Sekhon, B.S. and S.R. Kamboj, Inorganic nanomedicine—Part 1.
Nanomedicine: Nanotechnology, Biology and Medicine. 6(4): p. 516-522.
22. Fan, Z., Theranostic nanomedicine for cancer detection and treatment.
Journal of Food and Drug Analysis, 2014. 22(1): p. 3-17.
23. Monduzzi, M., From self-assembly fundamental knowledge to
nanomedicine developments. Advances in Colloid and Interface Science,
2014. 205: p. 48-67.
24. Loeve, S., B.B. Vincent, and F. Gazeau, Nanomedicine metaphors: From
war to care. Emergence of an oecological approach. Nano Today, 2013.
8(6): p. 560-565.
25. Jee, J.P., Cancer targeting strategies in nanomedicine: Design and
application of chitosan nanoparticles. Current Opinion in Solid State and
Materials Science, 2012. 16(6): p. 333-342.
26. Zarbin, A.J.G., Química de (nano)materiais. Química Nova, 2007. 30: p.
1469-1479.
27. Lee, J.D., Química inorgânica não tão concisa. 1999, Edgard Blücher
São Paulo.
28. V. J. Schacht, L.V.N., S. K. Sandhi, L. Chen, T. Henni, P. J. Klar, K.
Theophel, S. Schnell, M. Bunge, Effects of silver nanoparticles on
microbial growthdynamics. Journal of Applied Microbiology 2012. 114: p.
11.
29. Anstas PT, W.J., Green Chemistry: Theory and Practice. 1998, New York:
Oxford University Press, Inc.
30. DeSimone, J.M., Practical Approaches to Green Solvents. Science, 2002.
297: p. 5.
43
31. Gross, R.A. and B. Kalra, Biodegradable Polymers for the Environment.
Science, 2002. 297(5582): p. 803-807.
32. Poliakoff, M. and P. Anastas, A principled stance. Nature, 2001.
413(6853): p. 257-257.
33. Raveendran, P., J. Fu, and S.L. Wallen, Completely “Green” Synthesis
and Stabilization of Metal Nanoparticles. Journal of the American
Chemical Society, 2003. 125(46): p. 13940-13941.
34. Lenardão, E.J., "Green chemistry": os 12 princípios da química verde e
sua inserção nas atividades de ensino e pesquisa. Química Nova, 2003.
26: p. 123-129.
35. Sharma, V.K., R.A. Yngard, and Y. Lin, Silver nanoparticles: Green
synthesis and their antimicrobial activities. Advances in Colloid and
Interface Science, 2009. 145(1–2): p. 83-96.
36. Amanullah, M. and L. Yu, Environment friendly fluid loss additives to
protect the marine environment from the detrimental effect of mud
additives. Journal of Petroleum Science and Engineering, 2005. 48(3–4):
p. 199-208.
37. Yu, D. and V.W.W. Yam, Hydrothermal-Induced Assembly of Colloidal
Silver Spheres into Various Nanoparticles on the Basis of HTAB-Modified
Silver Mirror Reaction. The Journal of Physical Chemistry B, 2005.
109(12): p. 5497-5503.
38. Eustis, S., Growth and fragmentation of silver nanoparticles in their
synthesis with a fs laser and CW light by photo-sensitization with
benzophenone. Photochemical & Photobiological Sciences, 2005. 4(1): p.
154-159.
39. Sudeep, P.K. and P.V. Kamat, Photosensitized Growth of Silver
Nanoparticles under Visible Light Irradiation: A Mechanistic Investigation.
Chemistry of Materials, 2005. 17(22): p. 5404-5410.
44
40. Collera-Zúñiga, O., F. Garcıa Jiménez, and R. Meléndez Gordillo,
Comparative study of carotenoid composition in three mexican varieties of
Capsicum annuum L. Food Chemistry, 2005. 90(1–2): p. 109-114.
41. Jagadeesh, B.H., T.N. Prabha, and K. Srinivasan, Activities of β-
hexosaminidase and α-mannosidase during development and ripening of
bell capsicum (Capsicum annuum var. variata). Plant Science, 2004.
167(6): p. 1263-1271.
42. Weinstock, I.A., Homogeneous-Phase Electron-Transfer Reactions of
Polyoxometalates. [(Chem. Rev. 1998, 98, 113 (this issue). Published on
the Web January 22, 1998.]. Chemical Reviews, 1998. 98(1): p. 389-390.
43. Hill, C.L., Introduction: PolyoxometalatesMulticomponent Molecular
Vehicles To Probe Fundamental Issues and Practical Problems. Chemical
Reviews, 1998. 98(1): p. 1-2.
44. Zhang, G., Synthesis of various crystalline gold nanostructures in water:
The polyoxometalate [small beta]-[H4PMo12O40]3- as the reducing and
stabilizing agent. Journal of Materials Chemistry, 2009. 19(45): p. 8639-
8644.
45. Campbell, M.K., Bioquímica. Vol. 3. 2007, São Paulo: Thomson Learning.
46. Organization, W.H. Antimicrobial resistance. 2015 [cited 2015 Jun-7-
2015]; Available from:
http://www.who.int/mediacentre/factsheets/fs194/en/.
47. Tortorra, G.J., B.R. Funke, and C.L. Case, Microbiologia. 8 ed. 2005, Porto
Alegre: Artmed.
48. Heilman, S., Efeito da radiação ionizante nos revestimentos de cateteres
de poliuretano com nanopartículas de prata. 2015, IPEN - USP: São
Paulo. p. 90.
49. Kim, K.S., K.H. Jung, and S.-B. Jung, Design and fabrication of screen-
printed silver circuits for stretchable electronics. Microelectronic
Engineering, 2014. 120(Complete): p. 216-220.
45
50. Fukuda, K., Organic integrated circuits using room-temperature sintered
silver nanoparticles as printed electrodes. Organic Electronics, 2012.
13(12): p. 3296-3301.
51. Natsuki, J. and T. Abe, Synthesis of pure colloidal silver nanoparticles with
high electroconductivity for printed electronic circuits: The effect of amines
on their formation in aqueous media. Journal of Colloid and Interface
Science, 2011. 359(1): p. 19-23.
52. Hsieh, C.T., C. Pan, and W.Y. Chen, Synthesis of silver nanoparticles on
carbon papers for electrochemical catalysts. Journal of Power Sources,
2011. 196(15): p. 6055-6061.
53. Seo, J.H., Cytotoxicity of serum protein-adsorbed visible-light
photocatalytic Ag/AgBr/TiO2 nanoparticles. Journal of Hazardous
Materials, 2011. 198: p. 347-355.
54. Huang, Q., W. Shen, and W. Song, Synthesis of colourless silver precursor
ink for printing conductive patterns on silicon nitride substrates. Applied
Surface Science, 2012. 258(19): p. 7384-7388.
55. Zhou, X., Enhanced dispersibility and dispersion stability of
dodecylamine-protected silver nanoparticles by dodecanethiol for ink-jet
conductive inks. Applied Surface Science, 2014. 292: p. 537-543.
56. Kosmala, A., Synthesis of silver nano particles and fabrication of aqueous
Ag inks for inkjet printing. Materials Chemistry and Physics, 2011. 129(3):
p. 1075-1080.
57. Ataeefard, M. and S. Sharifi, Antibacterial flexographic ink containing silver
nanoparticles. Progress in Organic Coatings, 2014. 77(1): p. 118-123.
58. Said, J., An in vitro test of the efficacy of silver-containing wound
dressings against Staphylococcus aureus and Pseudomonas aeruginosa
in simulated wound fluid. International Journal of Pharmaceutics, 2014.
462(1–2): p. 123-128.
46
59. Gaisford, S., An in vitro method for the quantitative determination of the
antimicrobial efficacy of silver-containing wound dressings. International
Journal of Pharmaceutics, 2009. 366(1–2): p. 111-116.
60. Hebeish, A., Antimicrobial wound dressing and anti-inflammatory efficacy
of silver nanoparticles. International Journal of Biological Macromolecules,
2014. 65: p. 509-515.
61. Silver, S. and L.T. Phung, Bacterial heavy metal resistance: New
Surprises. Annual Review of Microbiology, 1996. 50(1): p. 753-789.
62. M, C., Antibacterial and bioactive silver-containing Na2OCaO2SiO2 glass
prepared by sol-gel method. Journal of Materials Science and Materials
for Medicine, 2004. 15: p. 7.
63. Crabtree, J., The efficacy of silver-ion implanted catheters in reducing
peritoneal dialysis-related infections. Peritoneal Dialysis International,
2003. 23(4): p. 368-374.
64. Zhao, G. and J.S. Stevens, Multiple parameters for the comprehensive
evaluation of the susceptibility of Escherichia coli to the silver ion. .
Biomaterials, 1998. 11: p. 6.
65. Aymonier, C., Hybrids of silver nanoparticles with amphiphilic
hyperbranched macromolecules exhibiting antimicrobial properties.
Chemical Communications, 2002(24): p. 3018-3019.
66. Ansari, M., Evaluation of antibacterial activity of silver nanoparticles
against MSSA and MRSA on isolates from skin infections. Biology and
Medicine, 2011.
67. Libor, K. and S. Jana, Comment on 'Preparation and antibacterial activity
of Fe 3 O 4 @Ag nanoparticles'. Nanotechnology, 2009. 20(2): p. 028001.
68. Rai, M., A. Yadav, and A. Gade, Silver nanoparticles as a new generation
of antimicrobials. Biotechnology Advances, 2009. 27(1): p. 76-83.
69. Lok, C.N., Proteomic Analysis of the Mode of Antibacterial Action of Silver
Nanoparticles. Journal of Proteome Research, 2006. 5(4): p. 916-924.
47
70. Kim, J.S., Antimicrobial effects of silver nanoparticles. Nanomedicine:
Nanotechnology, Biology and Medicine, 2007. 3(1): p. 95-101.
71. Magaña, S.M., Antibacterial activity of montmorillonites modified with
silver. Journal of Molecular Catalysis A: Chemical, 2008. 281(1–2): p. 192-
199.
72. Shin, S.H., The effects of nano-silver on the proliferation and cytokine
expression by peripheral blood mononuclear cells. International
Immunopharmacology, 2007. 7(13): p. 1813-1818.
73. Arjunan, N.K., Green Synthesis of Silver Nanoparticles for the Control of
Mosquito Vectors of Malaria, Filariasis, and Dengue. Vector-Borne and
Zoonotic Diseases, 2011. 12(3): p. 262-268.
74. Kim, T.N., Antimicrobial effects of metal ions (Ag+, Cu2+, Zn2+) in
hydroxyapatite. Journal of Materials Science: Materials in Medicine, 1998.
9(3): p. 129-134.
75. Cho, K.H., The study of antimicrobial activity and preservative effects of
nanosilver ingredient. Electrochimica Acta, 2005. 51(5): p. 956-960.
76. Meenal, K., Extracellular synthesis of silver nanoparticles by a silver-
tolerant yeast strain MKY3. Nanotechnology, 2003. 14(1): p. 95.
77. Souza, G.I.H., Utilization of Fusarium oxysporum in the biosynthesis of
silver nanoparticles and its antibacterial activities. , in Xth National Meeting
of Environmental Microbiology. 2004: Curitiba, PR (Brazil)
78. Durán, N., Mechanistic aspects of biosynthesis of silver nanoparticles by
several Fusarium oxysporum strains. Journal of Nanobiotechnology, 2005.
3: p. 8-8.
79. Wright, G.D., Resisting resistance: new chemical strategies for battling
superbugs. Chemistry & Biology, 2000. 7(6): p. R127-R132.
80. Mogensen, K.B. and K. Kneipp, Size-Dependent Shifts of Plasmon
Resonance in Silver Nanoparticle Films Using Controlled Dissolution:
48
Monitoring the Onset of Surface Screening Effects. The Journal of
Physical Chemistry C, 2014. 118(48): p. 28075-28083.
81. Mock, J.J., Shape effects in plasmon resonance of individual colloidal
silver nanoparticles. The Journal of Chemical Physics, 2002. 116(15): p.
6755-6759.
82. Malinsky, M.D., Chain Length Dependence and Sensing Capabilities of
the Localized Surface Plasmon Resonance of Silver Nanoparticles
Chemically Modified with Alkanethiol Self-Assembled Monolayers. Journal
of the American Chemical Society, 2001. 123(7): p. 1471-1482.
83. Sonnichsen, C., A molecular ruler based on plasmon coupling of single
gold and silver nanoparticles. Nat Biotech, 2005. 23(6): p. 741-745.
84. Duval Malinsky, M., Nanosphere Lithography: Effect of Substrate on the
Localized Surface Plasmon Resonance Spectrum of Silver Nanoparticles.
The Journal of Physical Chemistry B, 2001. 105(12): p. 2343-2350.
85. Melo, A.D.Q., Estudo da utilização de coacervatos de polifosfato de sódio
na obtenção de materiais com nanopartículas metálicas e magnéticas, in
Departamento de Química Orgânica e Inorgânica. 2011, Universidade
Federal do Ceará: Brasil. p. 89.
86. Santos, K.O., Nanopartículas de prata e prata-paládio estabilizadas pela
polietilenoimina linear funcionalizada: formação, caracterização e
aplicações catalíticas, in Departamento de Química. 2012, Universidade
Federal de Santa Catarina: Brasil. p. 153.
87. Vidhu, V.K., S.A. Aromal, and D. Philip, Green synthesis of silver
nanoparticles using Macrotyloma uniflorum. Spectrochimica Acta Part A:
Molecular and Biomolecular Spectroscopy, 2011. 83(1): p. 392-397.
88. Rastogi, L. and J. Arunachalam, Sunlight based irradiation strategy for
rapid green synthesis of highly stable silver nanoparticles using aqueous
garlic (Allium sativum) extract and their antibacterial potential. Materials
Chemistry and Physics, 2011. 129(1–2): p. 558-563.
49
89. Moldovan, B., A green approach to phytomediated synthesis of silver
nanoparticles using Sambucus nigra L. fruits extract and their antioxidant
activity. Journal of Molecular Liquids, 2016. 221: p. 271-278.
90. Espenti, C.S., K.S.V.K. Rao, and K.M. Rao, Bio-synthesis and
characterization of silver nanoparticles using Terminalia chebula leaf
extract and evaluation of its antimicrobial potential. Materials Letters,
2016. 174: p. 129-133.
91. Kumar, V.A., Synthesis of nanoparticles composed of silver and silver
chloride for a plasmonic photocatalyst using an extract from needles of
Pinus densiflora. Materials Letters, 2016. 176: p. 169-172.
92. Ramanibai, R. and K. Velayutham, Synthesis of silver nanoparticles using
3,5-di-t-butyl-4-hydroxyanisole from Cynodon dactylon against Aedes
aegypti and Culex quinquefasciatus. Journal of Asia-Pacific Entomology.
93. Ravichandran, V., Green synthesis of silver nanoparticles using
Atrocarpus altilis leaf extract and the study of their antimicrobial and
antioxidant activity. Materials Letters, 2016. 180: p. 264-267.
94. El-Sherbiny, I.M., A. El-Shibiny, and E. Salih, Photo-induced green
synthesis and antimicrobial efficacy of poly (ɛ-
caprolactone)/curcumin/grape leaf extract-silver hybrid nanoparticles.
Journal of Photochemistry and Photobiology B: Biology, 2016. 160: p. 355-
363.
95. El-Shamy, A.G., W. Attia, and K.M. Abd El-Kader, The optical and
mechanical properties of PVA-Ag nanocomposite films. Journal of Alloys
and Compounds, 2014. 590: p. 309-312.
96. Verma, D.K., S.H. Hasan, and R.M. Banik, Photo-catalyzed and phyto-
mediated rapid green synthesis of silver nanoparticles using herbal extract
of Salvinia molesta and its antimicrobial efficacy. Journal of
Photochemistry and Photobiology B: Biology, 2016. 155: p. 51-59.
50
97. Pal, A., S. Shah, and S. Devi, Microwave-assisted synthesis of silver
nanoparticles using ethanol as a reducing agent. Materials Chemistry and
Physics, 2009. 114(2–3): p. 530-532.
98. Yang, J. and J. Pan, Hydrothermal synthesis of silver nanoparticles by
sodium alginate and their applications in surface-enhanced Raman
scattering and catalysis. Acta Materialia, 2012. 60(12): p. 4753-4758.
99. Darroudi, M., Green synthesis of colloidal silver nanoparticles by
sonochemical method. Materials Letters, 2012. 66(1): p. 117-120.
100. Zhanjiang, Z. and L. Jinpei, Synthesis and Characterization of Silver
Nanoparticles by a Sonochemical Method. Rare Metal Materials and
Engineering, 2012. 41(10): p. 1700-1705.
101. Mandal, A., Synthesis, characterization and comparison of antimicrobial
activity of PEG/TritonX-100 capped silver nanoparticles on collagen
scaffold. Colloids and Surfaces B: Biointerfaces, 2012. 90: p. 191-196.
102. Wojtysiak, S. and A. Kudelski, Influence of oxygen on the process of
formation of silver nanoparticles during citrate/borohydride synthesis of
silver sols. Colloids and Surfaces A: Physicochemical and Engineering
Aspects, 2012. 410: p. 45-51.
103. Yan, J., Antibacterial activity of silver nanoparticles synthesized In-situ by
solution spraying onto cellulose. Carbohydrate Polymers, 2016. 147: p.
500-508.
104. Xu, L., Droplet synthesis of silver nanoparticles by a microfluidic device.
Chemical Engineering and Processing: Process Intensification, 2016. 102:
p. 186-193.
105. Kaur, J. and K. Tikoo, Evaluating cell specific cytotoxicity of differentially
charged silver nanoparticles. Food and Chemical Toxicology, 2013. 51: p.
1-14.
51
106. Yang, C., S. Jung, and H. Yi, A biofabrication approach for controlled
synthesis of silver nanoparticles with high catalytic and antibacterial
activities. Biochemical Engineering Journal, 2014. 89: p. 10-20.
107. Jia, C.J. and F. Schuth, Colloidal metal nanoparticles as a component of
designed catalyst. Physical Chemistry Chemical Physics, 2011. 13(7): p.
2457-2487.
108. Signori, A.M., Formation of Catalytic Silver Nanoparticles Supported on
Branched Polyethyleneimine Derivatives. Langmuir, 2010. 26(22): p.
17772-17779.
109. Gentry, S.T., S.J. Fredericks, and R. Krchnavek, Controlled Particle
Growth of Silver Sols through the Use of Hydroquinone as a Selective
Reducing Agent. Langmuir, 2009. 25(5): p. 2613-2621.
110. Aiken Iii, J.D. and R.G. Finke, A review of modern transition-metal
nanoclusters: their synthesis, characterization, and applications in
catalysis. Journal of Molecular Catalysis A: Chemical, 1999. 145(1–2): p.
1-44.
111. Patakfalvi, R., S. Papp, and I. Dékány, The kinetics of homogeneous
nucleation of silver nanoparticles stabilized by polymers. Journal of
Nanoparticle Research, 2007. 9(3): p. 353-364.
112. Bönnemann, H., Nanoscale colloidal metals and alloys stabilized by
solvents and surfactants Preparation and use as catalyst precursors.
Journal of Organometallic Chemistry, 1996. 520(1): p. 143-162.
113. SHABBEER, H.S., Effect of Acidic and Basic Conditions on the Plasmon
Band of Colloidal Silver. 2012, 2012. 9(3): p. 9.
114. Yan, Y., Capping effect of reducing agents and surfactants in
synthesizing silver nanoplates. Transactions of Nonferrous Metals Society
of China, 2014. 24(11): p. 3732-3738.
52
115. Lah, N.A.C. and M.R. Johan, Facile shape control synthesis and optical
properties of silver nanoparticles stabilized by Daxad 19 surfactant.
Applied Surface Science, 2011. 257(17): p. 7494-7500.
116. Hebeish, A., T.I. Shaheen, and M.E. El-Naggar, Solid state synthesis of
starch-capped silver nanoparticles. International Journal of Biological
Macromolecules, 2016. 87: p. 70-76.
117. Vanamudan, A. and P.P. Sudhakar, Biopolymer capped silver
nanoparticles with potential for multifaceted applications. International
Journal of Biological Macromolecules, 2016. 86: p. 262-268.
118. Barnes, W.L., A. Dereux, and T.W. Ebbesen, Surface plasmon
subwavelength optics. Nature, 2003. 424(6950): p. 824-830.
119. Wriedt, T., Mie theory 1908, on the mobile phone 2008. Journal of
Quantitative Spectroscopy & Radiative Transfer, 2008. 109: p. 6.
53
CHAPTER 2
GREEN SYNTHESIS OF SILVER NANOPARTICLES
TROUGH REDUCING SUGARS
54
CHAPTER 2 – GREEN SYNTHESIS OF SILVER NANOPARTICLES TROUGH
REDUCING SUGARS
1 INTRODUCTION
This chapter describes the synthesis of AgNP, such as the
characterization procedure and the preliminary tests with the nanoparticles. As
described on general introduction of this work, four sugars were used. Among
these sugars, only three of them were effectively continued because of the non-
needed use of NaOH to reduce silver ions, and the main stabilizer agent was
sodium citrate, because of surfactant properties of sodium dodecyl sulfate.
However, all the procedures are described.
2 EXPERIMENTAL
2.1 Materials
Were used to perform this synthesis silver nitrate (Dinâmica, São Paulo),
sodium citrate (Neon, São Paulo), sodium dodecyl sulfate (Dinâmica, São Paulo),
arabinose (Sigma Aldrich, São Paulo), glucose (Sigma Aldrich, São Paulo),
ribose (Sigma Aldrich, São Paulo), xylose (Dinâmica, São Paulo) and sodium
hidroxyde (Dinâmica, São Paulo).
2.2 Methods
500mL of 5.0mM silver nitrate solution was warmed to desired
temperature (described on Table 2.1). Once the temperature was achieved, it
was added to the system 1.00g of sugar and 0.50g of sodium citrate or sodium
dodecyl sulfate, according the desired synthesis.
After 10 minutes, it was possible to verify the permanent yellow color of
the solution, which is a visual sign of successful reaction. The solution is
transferred to a bottle and then stored at room temperature (25ºC to this work).
This method is shown at Figure 2.1. Each sample was then named according to
the Table 2.1.
55
Figure 2.1 – AgNP synthesis: illustrated steps (citrate series).
Source: author.
Table 2.1 – AgNPs samples and temperature of synthesis.
Sample Sugar Temperature
AgAC-01 Arabinose 60 ºC
AgAC-02 Arabinose 80 ºC
AgAC-03 Arabinose 100 ºC
AgRC-01 Ribose 60 ºC
AgRC-02 Ribose 80 ºC
AgRC-03 Ribose 100 ºC
AgXC-01 Xylose 50 ºC
AgXC-02 Xylose 60 ºC
AgXC-03 Xylose 70 ºC
AgXC-04 Xylose 80 ºC
AgXC-05 Xylose 90 ºC
AgXC-06 Xylose 100 ºC
Source: author.
The samples were labeled according to these terms: Ag is silver; A is
arabinose; C is citrate; R is ribose and X is xylose. The sequence 01-06 is
signaled to the temperature flow, from the smaller to higher (60, 80 and 100 are
respectively 1, 2 and 3 on ribose and arabinose, while in xylose 60, 70, 80, 90
and 100 are 1, 2, 3, 4, 5 and 6.
56
2.2.1 Concentrating AgNPs
Once the AgNPs were obtained, the solution was carried to a centrifuge
and then centrifuged for 20 minutes over 10000rpm. After two cycles of
centrifuge, the centrifuged liquid at the bottom of the tube is pipetted to another
bottle and stored under protection of light in a freezer at 5 ºC. Figure 2.2 shows
the before and after of centrifugation.
Figure 2.2 – Solution before (left) and after (right) centrifugation.
Source: author.
2.2.2 Preliminary antimicrobial assays
To verify the antimicrobial activity of AgNP, an in vitro assay was
performed against fungi of Aspergillus species, shown at Figure 2.3. In addition,
the samples chosen were the same of catalytic assays.
Figure 2.3 – Aspergillus fumigatus (a), A. terreus (b) and A. niger (c).
(a) (b) (c)
Source: author.
57
3 CHARACTERIZATION
3.1 UV-Vis spectroscopy
It was used a Thermo Scientific GENESYS 10s spectrophotometer to
characterize the obtaining of AgNPs, once these nanoparticles have
characteristics absorption bands – plasmon bands – at 420nm. The same
technique was performed to estimate the size of AgNP.
3.2 X-ray diffraction (XRD)
1.0mL of centrifuged AgNP was drop and dried into a glass plate and
then carried to a Phillips PW 1710 X-ray with copper tube K (k=1.54Ǻ) to verify
the obtaining of crystalline profile of silver.
3.3 Dynamic light scattering (DLS) and zeta potential
The dynamic light scattering technique was performed to obtain the
hydrodynamic radius of AgNPs, such as the zeta potential to evaluate the stability
of the nanoparticles in solution. It was used a Zetasizer NanoZS Malvern. It was
chosen the arabinose samples to represent the samples.
3.4 Scanning electron microscopy (SEM)
To investigate the morphology and topology properties of the AgNP, it
was performed a SEM assay. It was chosen the AgRC-01, AgRC-02 and AgRC-
03 samples to be characterized. The microscope was an electron microscope
Quanta-450 (FEI) with a field-emission gun (FEG), a 100 mm stage and an X-ray
detector (model 150, Oxford) for energy-dispersive X-ray spectroscopy (EDS),
from Central Analítica – Universidade Federal do Ceará.
58
4 RESULTS AND DISCUSSION
4.1 UV-Vis spectroscopy
This technique has shown itself as an excellent way to characterize the
production of AgNPs, once the characteristic band obtained near 420nm is proper
of the AgNP, and is also called plasmon band [1]. The Figures below illustrate
the absorption of AgNP solutions, according to the sugar used to synthesize the
nanoparticles. The Figures 2.4, 2.5 and 2.6 show the absorption of the AgNPs
solution. The first one shown is the AgNPs produced using arabinose as reducing
sugar.
Figure 2.4 – UV-Vis absorption of AgAC-01, AgAC-02 and AgAC-03.
Source: author.
For each one of the samples, the max were 410, 404 and 401nm for
AgAC-01, AgAC-02 and AgAC-03, respectively.
59
Then, it was performed the UV-Vis absorption of AgNP obtained by
synthesis using ribose, shown at Figure 2.5.
Figure 2.5 – UV-Vis absorption of AgRC-01, AgRC-02 and AgRC-03.
Source: author.
The profile of these nanoparticles looks uniform, when compared to the
previous (arabinose series). In addition, the bands are not as broad as the first
ones. This profile suggests that the ribose series gave a system of
monodispersive nanoparticles, where the size of the AgNPs is not significantly
different.
For each one of the samples, the max were 405, 399 and 398nm
respectively for AgRC-01, AgRC-02 and AgRC-03.
The last series, AgNPs obtained by xylose, is shown in Figure 2.6 below.
However, to this series it was performed a wide temperature range, exactly to
verify if there was any difference of synthesis among 50ºC and 100ºC. All
investigated temperature could synthesize AgNPs with discreet difference among
them. For each one of the samples, the max were 408, 408, 406, 402, 403 and
60
403nm for AgXC-01, AgXC-02, AgXC-03, AgXC-04, AgXC-05 and AgXC-06,
respectively
Figure 2.6 – UV-Vis absorption of AgXC-01, AgXC-02, AgXC-03, AgXC-04,
AgXC-05 and AgXC-06.
Source: author.
When it is observed the Figures of UV-Vis data, it is possible to
understand that the AgNP were obtained, once this region near 400nm works like
a real fingerprint to characterize silver nanoparticles [1-4]. Actually, there was not
significant band displacement, once all max were sufficient near (410 – 398) to
all samples. A complementary technique – XRD – will confirm the phases of
silver, as shown on next topic ahead (4.2).
61
As a general observation, the spectrum width of bands became smaller
with the increasing of temperature, which suggests that the systems, according
to the temperature rise, were getting more monodispersive, which has as
characteristic the uniform distribution of size of nanoparticles.
The solutions containing only the sugar without sodium citrate had shown
the same pale yellow aspect, however, when the UV-Vis spectra of these
samples are collected, there is no absorption bands, and it is also possible to
verify some remnants of solid at the bottom of bottle. Once the reaction occurs
with the oxidation of sugar and reduction of silver, it is possible to infer that the
AgNP are obtained, but without the capping agent they are not stable at solution.
The spectra data of the sugars without sodium citrate is shown in Figure 2.7,
where it is possible to see that the plasmon band is not present on those
solutions.
Figure 2.7 – UV-Vis specter of samples without sodium citrate.
Source: author.
62
It is also important to perform the UV-Vis spectra of silver nanoparticles
with sodium citrate, but without the reducing sugar, so it is possible to say, indeed,
that the sugars are the reactants that reduce silver under the investigated
conditions. Figure 2.8 shows the spectrum of silver with sodium citrate.
Figure 2.8 – UV-Vis specter of sample with sodium citrate only.
Source: author.
63
The UV-Vis spectra are particularly useful to determine the size of
nanoparticles. Slistan-Grijalva and co-workers [5, 6] elucidated that the particle
size could also be related with the intensity and full width half maximum (FWHM)
of the plasmon band. Schematically, it would be like this, shown at Figure 2.9:
Figure 2.9 – FWHM of an arbitrary measure.
Source: author.
As seen at Figure 2.9, there are two wavelengths highlighted in green:
370nm and 428nm. Both are related to the FWHM of this arbitrary absorption
band, which the maximum of absorption occurs with 0.595A (arbitrary unit). The
half is signaled to the both numbers already cited. So, when the difference of
them is taken, it is possible to obtain the half size of synthesized nanoparticles.
When applying this to the obtained nanoparticles, it is possible to catalog
the following values, presented at Table 2.2:
Table 2.2 – Particle mean size according FWHM.
64
Sample Particle medium size according FWHM
AgAC-01 89 nm
AgAC-02 67 nm
AgAC-03 58 nm
AgRC-01 73 nm
AgRC-02 60 nm
AgRC-03 60 nm
AgXC-01 73 nm
AgXC-02 70 nm
AgXC-03 69 nm
AgXC-04 61 nm
AgXC-05 60 nm
AgXC-06 55 nm
Source: author.
All the samples were affected by temperature variation. At higher values,
the system was able to synthesize smaller nanoparticles, which is an interesting
data. So, controlling the temperature – and keeping unchanged all the other
variables – it is possible to estimate the size distribution of silver nanoparticles,
according to the UV-Vis spectra.
This preliminary data is an estimative and needs another technique to
give it some robustness. Further, the dynamic light scattering and scanning
electron micrograph will complement this one, so the average size of obtained
silver nanoparticles may be effectively determined.
It is important to cite that Mohan and co-workers [7] produced
nanoparticles with an average size of 14 ± 4nm, starting from glucose as reducing
sugar, as well as Filippo and co-workers [8] obtained 5.2 nm particles for
saccharose and 60 nm for maltose.
65
4.2 XRD
In a way to complement the UV-Vis spectroscopy, XRD allows us to
confirm the obtained phases of the silver nanoparticles [4, 9-11]. Figure 2.10
shows the diffraction pattern for three AgNPs. The chosen samples were AgRC-
01, AgRC-02 and AgRC-03, which were synthesized from same sugar from
different temperatures.
Figure 2.10 – XRD pattern for AgRC-01, AgRC-02 and AgRC-03.
Source: author.
As verified at literature [82-84, 98], the diffractogram above allows to
conclude that silver is present (JCPDS-04-0783), which correspond to diffraction
angles in 38.22º, 44.4º, 64.5º and 77.46º. There was not significant influence of
temperature on the phases of the AgNP, once all three samples had shown
similar diffractograms.
66
4.3 DLS and zeta potential
This technique consists in the scattering of light, and allows us to know
the average hydrodynamic radius of particles. The data may be interpreted
according the following size parameters: by intensity, by volume and by number,
as well as intensity of diffusion. The first one gives the classes of size and the
average percentage relative, based on the intensity of light scattering. The size
by volume reveals the classes of size and average percentage relative based on
the volume occupied by the nanoparticles, while by number the data is revealed
as the number of nanoparticles. The last parameter is obtained from the intensity
of light scattering in solution.
Parallel to the parameters of interpretation of nanoparticles size, there is
the PDI, which indicates the polydispersion index. Monodispersive samples,
which indicate homogeneity of sizes, will show PDI values much smaller than 1
(maximum definite value). When there is proximity to unit, it is an indicative of a
polydispersive sample, and there is a great difference among the size of
nanoparticles, which leads the classification of the system to heterogeneous.
Figure 2.11 shows the size distribution by intensity of light scattering. The
values are also arranged into a Table (2.3).
Figure 2.11 – Size distribution by intensity of light scattering.
Source: author.
67
Table 2.3 – Size distribution by intensity of light scattering.
Sample PDI Size (nm) Int. (%) SD
AgXC-01 0.621 Peak 1 Peak 2 Peak 3
43.81 2.544 8.056
83.1 16.1 0.7
20.85 0.760 1.440
AgXC-02 0.539 Peak 1 Peak 2
27.29 1.898
80.4 19.6
11.04 0.4753
AgXC-03 0.525 Peak 1 Peak 2
32.49 1.945
80.9 19.1
13.98
0.6278
AgXC-04 0.467 Peak 1 Peak 2
29.66 1.907
87.6 12.4
11.46 0.4467
AgXC-05 0.459 Peak 1 Peak 2 Peak 3
28.9 2.182
0.8175
85.8 13.3 0.9
11.50
0.6385 0.1172
AgXC-06 0.389 Peak 1 Peak 2
96.36 5.441
99.2 0.8
62.26 1.170
Source: author.
Once the stoichiometric conditions of synthesis were the same,
temperature was the only factor that varied. Thus, the effect on the size and PDI
of system is related to range in temperature. According the temperature
increases, the PDI is systematically reduced, as can be observed at PDI 0.621 to
0.389. This fact suggests a tendency to a monodispersive system. However, the
data table shows an increasing on the particle size at the highest temperature,
but this same increase is observed at standard deviation at 100ºC. An interesting
data is acquired when the samples AgXC-04 and AgXC-05 are observed: the
values do not present a great range of changes, and this may be an indicative of
a range of temperature that has the same activation energy. Moreover, the UV-
Vis profiles of these two samples are almost the same (as shown at Figure 2.6).
To verify the stability of the system, it was perform the analyses of the
zeta potential. AgXC-05 and AgXC-06 were selected ones to be analyzed,
because of the two lowest polydispersion indexes.
68
When performed the zeta potential analyses, some data were obtained
and are shown at Figures 2.12 and 2.13 below.
Figure 2.12 – Zeta potential of AgXC-05 sample.
Source: author
Figure 2.13 – Zeta potential of AgXC-06 sample.
Source: author
69
The obtained values for the samples were -51.4mV and -41.4mV,
respectively. According to the following Table 2.4, the expected values for zeta
potential are [12, 13]:
Table 2.4 – Zeta potential values
Zeta potential (in mV) Effect on solution
0 to ±10mV Coagulation or flocculation
±10mV to ±30mV Instability
±30mV to ±40mV Moderate stability
±40mV to ±60mV Good stability
±60mV and further Excellent stability Source: Andrei [12] and Hanaor [13].
The negative value for the zeta potential is because of sodium citrate,
which has a liquid anionic charge of 3-. Still according to the previous table, the
obtained values lead to a solution with good stability, which may be attributed to
the sodium citrate stabilizing the silver nanoparticles.
70
4.4 SEM
Sajanlal and co-workers [14] had elucidated already that the morphology
and crystalline structure of nanoparticles influences also at the UV-Vis spectra,
so that the wavelength of absorption is function of these parameters of AgNP.
Knowing this, it is important to investigate the morphology and distribution
of silver nanoparticles to verify if there is a correlation between the shape and the
absorption spectrum. Thus, it was performed the SEM to investigate this
characteristic and also AgNPs size. As previous analysis, the chosen samples
were AgRC-01, AgRC-02 and AgRC-03.
To initiate the series, Figure 2.14a and 2.14b show particles obtained
from AgRC-01 sample.
Figure 2.14a – SEM images from AgRC-01 at 78,022x.
Source: author.
Even there are AgNP with different morphologies, such as prismatic,
cubic and rods, it is possible to verify that the predominant shape of nanoparticles
is spherical, which meets the UV-Vis specter. According to literature, plasmon
band around 400nm is characteristic of spherical nanoparticles.
71
Also, depending on the capping agent used and the conditions of
synthesis, it is possible to obtain different shapes of nanoparticles.
Figure 2.14b – SEM images from AgRC-01 at 424,973x.
Source: author.
This previous micrograph shows a nanoparticle which is the fusion of
another three ones. Clearly, it can be seen that these AgNPs are imperfect
spheres. This process occurs when the solid is coalescing, and the capping agent
did not work with efficacy to avoid the occurrence of coalescence.
The next images show the AgRC-02 samples that were investigated at
SEM.
72
Figure 2.15a – SEM images for AgRC-02 at 100,000x.
Source: author.
Figure 2.15b – SEM images for AgRC-02 at 187,802x.
Source: author.
73
It was possible to see the difference between the first and second
samples. While the first were nanoparticles with a definite shape, even if not
predominant, the AgRC-02 nanoparticles showed a higher tendency to coalesce,
although they were smaller than AgRC-01.
Finally, the AgRC-03 series had images that reveal well-definite spheres
and a weak tendency to coalesce, as can be seen at Figures 2.16a and 2.16b.
Figure 2.16a – SEM images for AgRC-03 at 100,000x.
Source: author.
It is possible to see that the AgNPs of AgRC-03 series are predominantly
spheres and separated each from other. The absence of agglomerates is a
remarkable characteristic of this, and might be an effect of temperature, once the
conditions of synthesis were the same to all samples of this series.
This region of SEM was expanded to 200,000x to better investigate the
shape and morphology of obtained silver nanoparticles. The result is shown at
Figure 2.16b.
74
Figure 2.16b - SEM images for AgRC-03 at 200,000x.
Source: author.
The AgNPs had coalesced in a discreet way, as can be seen at this
expanded micrograph. At the magnification the spheres are better perceived,
which leads to infer that at 100ºC and the other conditions, the predominant
shape will be spherical and the particles will be better spread.
Once the micrographs are taken, it is also possible to build a histogram
for the analyzed AgNPs, and then understand the distribution of size and its
average according to each sample. The Figures below show the histograms
based on particles count and sizes for the referred samples.
75
Figure 2.17 – Histogram of AgRC-01 (a), AgRC-02 (b) and AgRC-03 (c) samples.
(a) (b)
(c)
Source: author
The histograms showed a tendency of system to obtain smaller nanoparticles when the temperature increases. This may be
attributed to the faster formation of nanoparticles and consequently stabilization with the citrate ion, which avoid the agglomeration of
the grains and lead to a more monodispersive and definite system.
76
4.5 Antimicrobial activity
When observing the results of the antimicrobial assay, it can be seen that
all the three nanoparticles had shown some results in a way to avoid the growth
of fungi. The Figures below show the behavior of the species when exposed to
the AgNP.
Figure 2.18 – Aspergillus fumigatus: control (a), with AgRC-01 (b), AgRC-02 (c)
and AgRC-03 (d).
(a) (b)
(c) (d)
Source: author.
77
Figure 2.19 – Aspergillus terreus: control (a), with AgRC-01 (b), AgRC-02 (c) e
AgRC-03 (d).
(a) (b)
(c) (d)
Source: author.
78
Figure 2.20 – Aspergillus niger: control (a), with AgRC-01 (b), AgRC-02 (c) e
AgRC-03 (d).
(a) (b)
(c) (d)
Source: author.
79
The images give interesting results. At the first study, the nanoparticles
had progressively slowed the growing of fungi, and AgRC-03 showed the highest
effectiveness in this process. This effect is easily seen to A. niger and A.
fumigatus species, which clearly the fungi are smaller when compared to the
control strain.
The specie A. terreus presented a different behavior: apparently there was
a stimulus to the growing of fungus. However, when it is better observed, it is
possible to verify that there was a kind of section, as the fungus was constantly
sliced, and could not grow as one.
The Figures and Tables below brings graphics and data the show that the
growth profile of fungi along 3 and 6 days exposed to AgNP. The results
illustrated leads us to believe that the AgNP synthesized at 100ºC show better
inhibitory properties over the others, which also leads us to assume that the
temperature of synthesis is an important factor to consider when AgNP are about
to be obtained.
The Figures show the size – Y axis – in millimeters and the counting days
– X axis.
80
Figure 2.21 – Growing of A. niger along six days.
Source: author.
Table 2.5 – Growing of A. niger versus time
A. niger (days) Control (mm) AgRC-01 (mm) AgRC-02 (mm) AgRC-03 (mm)
1 - - - -
2 - - - -
3 60 60 50 40
4 68 60 60 48
5 80 80 70 50
6 80 80 80 60
Source: author.
81
Figure 2.22 – Growing of A. terreus along six days.
Source: author.
Table 2.6 – Growing A. terreus versus time.
A. terreus (days) Control (mm) AgRC-01 (mm) AgRC-02 (mm) AgRC-03 (mm)
1 - - - -
2 - - - -
3 60 60 50 40
4 68 60 60 48
5 80 80 70 50
6 80 80 80 60
Source: author.
82
Figure 2.23 – Growing of A. fumigatus along six days.
Source: author.
Table 2.7 – Growing of A. fumigatus versus time.
A. fumigatus (days) Controle (mm) AgRC-01 (mm) AgRC-02 (mm) AgRC-03 (mm)
1 - - - -
2 - - - -
3 35 35 27 15
4 50 40 38 16
5 55 50 42 25
6 65 54 48 28
Source: author.
These results point to a preliminary understanding that the AgNP show
antifungal activity, which opened way to perform other tests with another
microorganisms and also with another silver nanoparticles, obtained by different
synthesis and sugars, as well as different capping agents.
83
5 CONCLUSIONS
The primary target, obtaining of silver nanoparticles trough green
synthesis, was completely satisfactory. All the investigated sugars yield AgNPs
in all temperatures and were also stabilized by sodium citrate in all cases.
There is no remarkable differences among the obtained silver
nanoparticles, once they are characterized trough UV-Vis and showed absorption
in very close regions, which suggests that, as the max is function of morphology
of AgNPs, all the samples showed predominance of spherical shapes, which
indicates that the used sugars did not differ one from each other when the
synthesis looks for different shapes of nanoparticles.
Temperature had shown an important factor on the avoiding of
coalescence and definition of most symmetric spherical nanoparticles.
Another factor that was influenced by temperature was also the size of
nanoparticles, as DLS technique had shown. The performed techniques showed
that it was possible to obtain nanoparticles with different sizes and distributions.
The first results of antifungal use of AgNPs were quite satisfactory and
promising for further studies.
84
REFERENCES
1. Rastegarzadeh, S. and F. Hashemi, A surface plasmon resonance
sensing method for determining captopril based on in situ formation of
silver nanoparticles using ascorbic acid. Spectrochimica Acta Part A:
Molecular and Biomolecular Spectroscopy, 2014. 122: p. 536-541.
2. Liu, P., Fluorescence enhancement of quercetin complexes by silver
nanoparticles and its analytical application. Spectrochimica Acta Part A:
Molecular and Biomolecular Spectroscopy, 2014. 122: p. 238-245.
3. Chhatre, A., Color and surface plasmon effects in nanoparticle systems:
Case of silver nanoparticles prepared by microemulsion route. Colloids
and Surfaces A: Physicochemical and Engineering Aspects, 2012. 404: p.
83-92.
4. Bindhu, M.R. and M. Umadevi, Surface plasmon resonance optical sensor
and antibacterial activities of biosynthesized silver nanoparticles.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy,
2014. 121: p. 596-604.
5. Slistan-Grijalva, A., Assessment of growth of silver nanoparticles
synthesized from an ethylene glycol–silver nitrate–polyvinylpyrrolidone
solution. Physica E: Low-dimensional Systems and Nanostructures, 2005.
25(4): p. 438-448.
6. Slistan-Grijalva, A., Classical theoretical characterization of the surface
plasmon absorption band for silver spherical nanoparticles suspended in
water and ethylene glycol. Physica E: Low-dimensional Systems and
Nanostructures, 2005. 27(1–2): p. 104-112.
7. Mohan, S., Completely green synthesis of dextrose reduced silver
nanoparticles, its antimicrobial and sensing properties. Carbohydrate
Polymers, 2014. 106: p. 469-474.
85
8. Filippo, E., Green synthesis of silver nanoparticles with sucrose and
maltose: Morphological and structural characterization. Journal of Non-
Crystalline Solids, 2010. 356(6–8): p. 344-350.
9. Kumar, D., V. Palanichamy, and S. Roopan, Green synthesis of silver
nanoparticles using Alternantheradentata leaf extract at room temperature
and their antimicrobial activity. Spectrochim Acta A, 2014. 127: p. 4.
10. Ahluwalia, V., Green synthesis of silver nanoparticles by Trichoderma
harzianum and their bio-efficacy evaluation against Staphylococcus
aureus and Klebsiella pneumonia. Industrial Crops and Products, 2014.
55: p. 202-206.
11. Gade, A., Green synthesis of silver nanoparticles by Phoma glomerata.
Micron, 2014. 59: p. 52-59.
12. Chapter 5. Electroacoustic theory, in Studies in Interface Science, S.D.
Andrei and J.G. Philip, Editors. 2002, Elsevier. p. 153-179.
13. Hanaor, D., The effects of carboxylic acids on the aqueous dispersion
and electrophoretic deposition of ZrO2. Journal of the European Ceramic
Society, 2012. 32(1): p. 235-244.
14. Panikkanvalappil R. Sajanlal, et al. Anisotropic nanomaterials: structure,
growth, assembly, and functions. 2011 [cited 2016 5/13/2016]; Available
from:
http://www.nanoreviewsexperiments.net/index.php/nano/article/view/588
3/7100.
86
CHAPTER 3
ANTIFUNGAL ACTIVITY OF SILVER NANOPARTICLES OBTAINED BY
GREEN SYNTHESIS .
Published in mar/2015 Revista do Instituto de Medicina Tropical
Impact factor (2015): 1.114
Qualis CAPES Química: B3
Citations (until may/2016): 3 scopus; 3 web of science
87
CHAPTER 3 – ANTIFUNGAL ACTIVITY OF SILVER NANOPARTICLES
OBTAINED BY GREEN SYNTHESIS
1 INTRODUCTION
Silver nanoparticles (AgNPs) are a new material that has several
applications, they are used as sensors, catalysts, anticancer and antimicrobial.
AgNPs presented activity against bacteria, fungi and viruses [1]. However the
synthesis of AgNPs produces toxic waste that may affect human health and the
environment [2]. The green synthesis of AgNPs has used various ways. Among
them have plants, microorganisms and non-toxic substances [3-5].
Antimicrobial resistance is a global concern and has become a public
health problem. The emergence of new agents with activity against multiresistant
microorganisms is slow and due to this many patients die every year [6]. So,
nanotechnology can be an important tool in combating these important
pathogens [2].
Candida albicans and Candida tropicalis yeasts are responsible for a
number of important diseases and recently cases of resistance of the yeast to the
main antifungal have been described. New substances should be sought as an
alternative to combat the spread of resistance [7, 8]. C. albicans and C. tropicalis
are the main yeasts isolated from samples of patients admitted to Hospitals of
Ceara [9].
The aim of this study was to synthesize and caracterize silver
nanoparticles using glucose and ribose sugars as reducing agents and citrate as
the capping agent. The antifungal activity of the synthesized AgNPs was
evaluated against strains of C. albicans and C. tropicalis.
88
2 EXPERIMENTAL
The synthesis of AgNPs was performed using ribose. Briefly, 500mL of a
5.0mM AgNO3 solution was warmed to 50ºC and then added 1.00 g of ribose and
0.5 g of SDS (sodium dodecyl sulfate) as stabilizer. The SDS has the function of
preventing agglomeration and subsequent precipitation of AgNPs. The reaction
was considered complete when the solution acquired a pale yellow color,
characteristic of AgNPs [10, 11].
In this study 30 strains of Candida sp. were selected (14 C. albicans and
16 C. tropicalis). These yeasts were isolated from blood samples of patients
hospitalized in Ceará. Briefly, C. albicans was purified and identified on
chromogenic medium trough production of chlamydospores in rice extract agar
with Tween-80, and germ tube formation, as shown at Figure 3.1.
Figure 3.1 – Identification of C. tropicalis (a) and C. albicans (b) on chromogenic
medium.
(a)
(b)
Source: author.
The sensibility of Candida sp. was evaluated by well-diffusion method on
Mueller-Hinton medium supplemented with 2.0% glucose and 0.05% methylene
blue. The wells contained 80g AgNPs and placed on medium containing
Candida sp. The discs of amphotericin B 10g were used as control. The plates
89
were incubated for 35°C/24h, and after this period the halos were measured
(mm). Each test was conducted three times, according to the protocol of CLSI
M44-A2 [12, 13]. Confirmation of identification was carried out by molecular
biology.
3 CHARACTERIZATION
The purification was carried out by centrifugation at 10.000rpm during 10
minutes and the characterization of the synthesized AgNPs was performed using
an UV-Visible spectrophotometer (Thermo Scientific GENESYS™ 10s), by
scanning the absorbance spectra in 300-700 nm range of wavelength. The size
of AgNPs was analyzed on Zetasizer NanoZS Malvern® by dynamic light
scattering (DLS) [14].
4 RESULTS AND DISCUSSION
The production of AgNPs using ribose as a reducing agent and SDS
capping agent was simple and easy to perform. The whole process is completed
in 30 min. The solution of AgNPs had a pale yellow as shown in Figure 3.2.
Figure 3.2 – AgNP solution.
Source: author.
The chemical reaction proposed can be represented by the equation
below:
C5H10O5(aq) + 2Ag+(aq) C5H8O5(aq) + 2H+
(aq) + 2Ag(s) (1)
90
The synthesized AgNPs showed strong spectrophotometric absorption
around 420nm as shown in Figure 3.3.
Figure 3.3 – UV-Vis specter of AgNP.
Source: author.
This is a typical behavior of these structures. SDS as the capping agent
provided prolonged stability at up to 4 months when stored at room temperature
and exposed to ambient light. Some sugars have reducing properties and are
used in the production of nanoparticles, once the process does not harm the
environment because it does not produce toxic waste, and require no accelerator
[15].
Nanotechnology is a new area that study particles which has size up to
100 nm, and the AgNPs produced in this work had size of 12.49 nm, as shown in
Figure 3.4 below. In a work using glucose as reducing agent [16], the size of
AgNPs was around 15 nm.
Figure 3.4 – Size distribution by volume (DLS).
Source: author.
91
The AgNPs exhibit high antimicrobial activity and this property can be
very useful, especially against microorganisms resistant to conventional
antimicrobial [17]. The effect is numerically shown at Table 3.1.
Table 3.1 – Effect of AgNPs produced by green synthesis and Amphotericin B
against C. albicans and C. tropicalis.
AgNPs Amphotericin
B
Strains (n) Range (mm) Halo (mm) Range (mm) Halo (mm)
(Media+/-SD) Media+/-SD) Media±SD Media±SD
C. albicans (14) 17-30 23±4 15-25 20±3
C. tropicalis (16) 12-30 21±4 15-25 20±3
Source: author
It is possible to extract from results that the halos of inhibition showed a
mean near medium among the used AgNP and the amphotericin B. Only the
standard deviation of the AgNP were higher than the control’s drug one.
Statistically, there are no significant differences among them – AgNP – but when
compared to the control drug, it is possible to see that the activity was clearly
higher and significant, because p < 0.05.
These results are kind relevant, once C. albicans is the most isolated
specie in Ceará, and C. tropicalis the second one [9]. So, the AgNP open way to
further studies that may clear the elucidation of the mechanism and suggest new
alternatives to combat fungal infections, especially in immunodeficient patients.
92
C. albicans and C. tropicalis showed high sensitivity AgNPs, these
particles showed fungicidal activity as can be seen in Figure 3.5.
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).
Source: author.
The activity of 80 g of AgNPs in this study can be compared with the
activity of amphotericin B, a powerful antifungal (Table 2.1). Studies highlight the
activity with activity of AgNPs against Candida spp [11, 18]. Strains of C. albicans
were more sensitive the AgNPs than C. tropicalis, as can be seen in Table 3.1.
The results of the performed test were very good, once it is visually
possible to see a higher effectiveness action of AgNP when compared to the
control drug. The halos may be identified at previous Figure when we observe
the circle regions where there was no fungal growth, what indicates that a
satisfactory inhibition of growing is present. Even its not described on this work,
it was used AgNP obtained by glucose as preliminary tests, and these
nanoparticles had shown activity as well. These results corroborate with literature
data [19, 20], which the AgNP show effective antimicrobial activity, turning
themselves into a good choice to avoid these microorganisms.
It is known that the antifungal may be classified as fungistatic or
fungicides, which the first inhibits the growing while the second kills the
93
microorganism. Amphotericin B is a fungistatic one. It can be seen that the
nanoparticles act like a fungicide agent. However, the mechanism of action of the
AgNP is not yet clear. Even under these circumstances, the AgNP may be used
to kill microorganism because they do not induce them to develop mechanisms
of resistance. Hence, it is a very interesting propose to use AgNP in new
products, such as wound heals, cosmetics, surgery materials and clothing, once
it avoid the growing of microorganisms by killing them.
Pant and coworkers [21] performed a study and verified that silver
nanoparticles shown activity against C. albicans and C. parapsilosis strains. An
interesting data is that the activity did not get higher with the raising of AgNP
concentration.
5 CONCLUSION
In conclusion, AgNPs were easily prepared and showed high activity
against C. albicans and C. tropicalis, similar activity observed by antifungal
amphotericin B and may represent an alternative for treating fungal infections.
94
REFERENCES
1. Kashyap, P.L., Myconanotechnology in agriculture: a perspective.
World Journal of Microbiology and Biotechnology, 2013. 29(2): p. 191-
207.
2. Shenashen, M.A., S.A. El-Safty, and E.A. Elshehy, Synthesis,
Morphological Control, and Properties of Silver Nanoparticles in Potential
Applications. Particle & Particle Systems Characterization, 2014. 31(3):
p. 293-316.
3. Iravani, S., Green synthesis of metal nanoparticles using plants. Green
Chemistry, 2011. 13(10): p. 2638-2650.
4. Sintubin, L., W. Verstraete, and N. Boon, Biologically produced
nanosilver: Current state and future perspectives. Biotechnology and
Bioengineering, 2012. 109(10): p. 2422-2436.
5. Quester, K., M. Avalos-Borja, and E. Castro-Longoria, Biosynthesis and
microscopic study of metallic nanoparticles. Micron, 2013. 54–55: p. 1-
27.
6. Barie, P.S., Multidrug-Resistant Organisms and Antibiotic Management.
Surgical Clinics of North America, 2012. 92(2): p. 345-391.
7. Cornistein, W., <i>Candida:</i> epidemiología y factores de riesgo para
especies no <i>albicans</i>. Enfermedades Infecciosas y Microbiología
Clínica, 2013. 31(06): p. 380-384.
8. Lockhart, S.R., Species Identification and Antifungal Susceptibility
Testing of Candida Bloodstream Isolates from Population-Based
Surveillance Studies in Two U.S. Cities from 2008 to 2011. Journal of
Clinical Microbiology, 2012. 50(11): p. 3435-3442.
9. Menezes, E.A., M.C.S.O. Cunha, and F.A. Cunha, Identificação
preliminar de algumas espécies do gênero Candida spp. em meio
cromógeno: resultados de dois anos de um estudo multicêntrico
realizado no Ceará. 2012, 2012. 40(4): p. 7.
95
10. Bhaduri, G.A., Green synthesis of silver nanoparticles using sunlight.
Journal of Photochemistry and Photobiology A: Chemistry, 2013. 258: p.
1-9.
11. Panáček, A., Antifungal activity of silver nanoparticles against Candida
spp. Biomaterials, 2009. 30(31): p. 6333-6340.
12. Institute., C.a.L.S., Method for antifungal disk diffusion susceptibility
testing of yeasts: approved standard M44-A2. 2008, Wayne.
13. Júnior A. A. V., M.E.A., Cunha F. A., Cunha M. C. S. O., Braz B. H. L.,
Capelo L. G., Silva C. L. F.. Comparação entre microdiluição e disco
difusão para o teste de susceptibilidade aos antifúngicos contra Candida
spp. Semina: Ciências Biológicas e da Saúde, 2012. 33(1): p. 8.
14. Gao, X., Green synthesis and characteristic of core-shell structure
silver/starch nanoparticles. Materials Letters, 2011. 65(19–20): p. 2963-
2965.
15. Oluwafemi, O.S., A facile completely ‘green’ size tunable synthesis of
maltose-reduced silver nanoparticles without the use of any accelerator.
Colloids and Surfaces B: Biointerfaces, 2013. 102: p. 718-723.
16. Amrute S. Lanje, S.J.S., Ramchandra B. Pode, Synthesis of silver
nanoparticles: a safer alternative to conventional antimicrobial and
antibacterial agents. Journal of Chemical and Pharmaceutical Research,
2010. 2(3): p. 6.
17. Sharma, V.K., R.A. Yngard, and Y. Lin, Silver nanoparticles: Green
synthesis and their antimicrobial activities. Advances in Colloid and
Interface Science, 2009. 145(1–2): p. 83-96.
18. Kumar, P., S. Senthamil Selvi, and M. Govindaraju, Seaweed-mediated
biosynthesis of silver nanoparticles using Gracilaria corticata for its
antifungal activity against Candida spp. Applied Nanoscience, 2013. 3(6):
p. 495-500.
96
19. Ghosh, S., Antimicrobial activity of highly stable silver nanoparticles
embedded in agar–agar matrix as a thin film. Carbohydrate Research,
2010. 345(15): p. 2220-2227.
20. Rai, M., Nanobiotecnologia verde: biossínteses de nanopartículas
metálicas e suas aplicações como nanoantimicrobianos. Ciência e
Cultura, 2013. 65: p. 44-48.
21. Pant, G., N. Nayak, and R. Gyana Prasuna, Enhancement of
antidandruff activity of shampoo by biosynthesized silver nanoparticles
from Solanum trilobatum plant leaf. Applied Nanoscience, 2013. 3(5): p.
431-439.
97
CHAPTER 4
ANTIFUNGAL ACTIVITY OF NANOBIOCOMPOSITE FILMS BASED IN
SILVER NANOPARTICLES OBTAINED BY GREEN SYNTHESIS
98
ABSTRACT
Agar films containing silver nanoparticles (AgNPs) are materials with many
applications, especially in food and pharmaceutical industry. In this work, it was
produced AgNPs by green synthesis, supported into a polymer matrix (Agar film,
AgFilm) and evaluated the antifungal activity. The AgNPs and AgFilm were
characterized using Ultraviolet-visible (UV-vis) spectroscopy, Fourier transform
infrared (FTIR), Dynamic Light Scattering (DLS), Powder X-ray Diffraction
(PXRD), Scanning Electron Microscopy (SEM) and Atomic Force Microscopic
(AFM). The AgNPs and AgFilm showed bands at 415 nm and 413 nm,
respectively. The diameter of AgNPs dispersed in AgFilm and thickness of the
film was 76.39 ± 41.86 nm and 35.4 ± 3.0 µM (mean ± SD), respectively. The
antifungal activity of AgFilm was evaluated against 20 strains of Candida
albicans. It was observed a high antifungal activity against C. albicans with high
values for zone of inhibition 19.1mm ± 1.8mm (Mean ± SD). This
nanobicomposite film can be a valuable ally in the treatment of superficial
infections caused by C. albicans.
Keywords: Silver nanoparticles; Agar film; Polymer composite; Antifungal
activity.
99
CHAPTER 4 – ANTIFUNGAL ACTIVITY OF NANOBIOCOMPOSITE FILMS
BASED IN SILVER NANOPARTICLES OBTAINED BY GREEN SYNTHESIS
1 INTRODUCTION
New materials, such as those obtained from polymer films, show
incorporating metal nanoparticles in their arrays. As a result, it is possible to
design a new product with many different applications: food, chemical and
biomedical industries [1, 2]. The raw material for the preparation of commercial
polymer films consists of petroleum oil, which is a non-renewable source. Due to
it, the interest in the use of natural polymers has increased, among which we can
mention the agar, gelatin, alginate, pullulan, starch, chitosan and cellulose [3, 4].
Several nanoparticles can be associated with polymer films, but stand
out silver nanoparticles (AgNPs) [5, 6], whose applications are numerous; healing
dressings, intravenous and urinary catheters, sutures, orthodontic adhesives,
surgical masks and films for use in the manufacturing industry, pharmaceutical
and food [7, 8]. Among the various polymers that are associated with AgNPs, one
of the most used is the agar [9, 10]. Agar is a complex polysaccharide present in
the cellular wall of red algae, namely, agarophytes [11]. The films containing
dispersed AgNPs in agar matrix are a subject that arouses interest due to its
antimicrobial and mechanical properties [12, 13].
Skin infections are very common in medical practice and the main
causative agents are fungi and highlight is given to Candida albicans [14]. These
polymer films with AgNPs in its constitution can be an alternative to combat the
skin infections caused by fungi as C. albicans [15]. Thus, the aim of this study
was to produce AgNPs by a non-toxic methodology and incorporate them into an
agar film to obtain a nanobiocomposite with antifungal activity against C. albicans.
100
2 EXPERIMENTAL
2.1 Materials
Silver nitrate (Dinâmica - São Paulo - Brasil, purity> 99%), sorbitol (Sigma
USA - purity-98%), agarose (Bio Basic – Canada - purity> 98%), sodium dodecyl
sulfate (SDS) (Vetec – Brazil - purity> 98%), NaOH (Dinâmica – Brazil -purity>
98%) were commercial products with analytical grade and used without any
purification.
2.2 AgNPs and AgFilm fabrication
The AgNPs were produced using glucose as reducing agent and SDS as
stabilizer. 1.0g of glucose, 0.5g of SDS and 500.0 mL of an aqueous solution of
AgNO3 5 mM were used for production of AgNPs. The method can be described
briefly: the mixture was maintained at 50 °C for 1 hour with constant stirring and
to accelerate the reaction, it was added 1.0 mL of NaOH 0.2 mol/L. The reaction
was considered complete when the solution acquired a yellow color characteristic
of the colloidal suspension with AgNPs. The Agfilm was obtained after adding 1.5
g of agarose and 0.5 g of sorbitol in 100.0 mL of the AgNPs suspension at 80 °C
for 1 hour with constant stirring. After this step, the solution was placed in plastic
plates. The plates were placed in an oven at 60 °C to complete the drying
process. AgFilms were removed from the plates and then conducted tests of
characterization and antifungal activity [9].
3 CHARACTERIZATION
3.1 Ultraviolet-visible (UV-Vis) spectroscopy
To confirm the production of AgNPs, the obtained solution and the AgFilm
were subjected to UV-vis spectroscopy in the range of 300-700 nm in a
spectrophotometer Thermo Scientific GENESYS ™ 10s.
3.2 Size
The average size of the AgNPs was measured in the colloidal solutions
using dynamic light scattering (DLS), a non-invasive backscatter technology. The
measurements were realized at a Zetasizer Nano ZS, Malvern Instruments-UK.
3.3 Fourier transform infrared (FT-IR) spectroscopy
101
The FTIR spectroscopy measurement of AgNPs and AgFilm powder
taken in KBr pellet was carried out using a Perkin-Elmer FT-IR Spectrum
ONE. The spectra were recorded in the wavenumber region of 4000-750 cm-1 at
resolution setting of 4 cm-1.
3.4 Powder X-ray diffraction (PXRD)
The AgNPs and AgFilm were crushed and placed in a glass support 2.5
cm x 2.5 cm and the diffractogram was measured using Cu-Ka radiation and
nickel with an energy of 40 kV and 30 mA with scanning 2θ 30°-80°. The samples
were analyzed in the PAN analytical X-pert Pro MRD (Amsterdam, Netherlands).
3.5 AgFilm thickness
The film thickness was measured using a digital caliper (Dial Thickness
gauge 7301, Mitutoyo, Tokyo, Japan) with accuracy of 0.01 mm. The
measurements were performed in 20 different parts of the AgFilm.
3.6 Microscopic features
Scanning electron microscopy (SEM) was carried out in the electron
microscope Quanta-450 (FEI) with a field-emission gun (FEG), a 100 mm stage
and an X-ray detector (model 150, Oxford) for energy-dispersive X-ray
spectroscopy (EDS). The Atomic Force Microscopy procedures were performed
using a Multimode Nanoscope IIIa (Bruker, CA, USA), in Tapping Mode (or
intermittent mode). In this scanning mode, the AFM tip touches the sample
surface with beats of similar amplitude to the resonance amplitude of the AFM
cantilever. It was used a rectangular probe model TESPA7 (Veeco) with nominal
spring constant of k= 20-80 N/m and resonance frequency of 291-326 kHz. The
scan parameters for the nanoparticles images acquisition were 0.5 Hz of scan
rate and 512 x 512 lines of resolution.
102
3.7 Antifungal activity of AgFilm
It were used 20 strains of Candida albicans isolated from patients treated
in public hospitals of the city of Fortaleza, Ceará, Brazil. To assess the antifungal
activity of the AgFilm it was used the method of diffusion in agar solid medium
Muller-Hinton supplemented with 2% glucose and 0.5 ug/mL of methylene blue.
AgFilm discs were cut to 10 mm in diameter. The solutions of C. albicans were
prepared and seeded in culture media and then placed the film disks. The plates
were incubated for a period of 24h to 35 °C. After this time, reading the inhibition
zones of fungal growth caused by the discs were qualitative and quantitative
analyzed. The growth inhibition zone was measured from disk center to the edge
at which fungal growth did not occur [16]. As control were used disks of film in
which no contained no AgNPs.
4 RESULTS AND DISCUSSION
Glucose is an non-toxic and inexpensive reagent sometimes used as
reduction agent in the reaction for obtaining AgNPs [17]. The synthesis conditions
of AgNPs can vary and incorporate other elements. In our study, it was used SDS
as a stabilizer, sodium hydroxide as accelerator, and sorbitol to provide elasticity
to AgFilm [18, 19]. The inclusion of AgNPs into the film was performed by the ex-
situ method, which consists in adding the as-prepared AgNPs to agar. AgNPs
bind to the polymer generating a nanostructured film with important biological
property. When AgNPs are supported within the polymer structure, the ratio of
aggregation forces between AgNPs and the polymer fibers are modified, Van der
Waals, electrostatic and magnetic forces now act not only in AgNPs: the polymer
structure also takes part in this power contest, and this factor may explain the
aggregation of AgNPs [20].
The AgFilm showed uniform appearance – as can be seen at Figure 4.1
– and presented thickness of 35.4 ± 3.0 µm (mean ± SD). Agar films described in
the literature have similar thickness, which enables its technological applications
[2, 9]. The placement of sorbitol in the film preparation protocol has the purpose
of giving plasticity and strength. The agar films described in the literature do not
add sorbitol, which make them brittle and difficult to handle [16].
103
Figure 4.1 – AgFilm.
Source: author.
Figure 4.2 shows UV-visible absorption spectra for AgNPs colloidal
suspension and AgFilm. The color suspension (yellow) and film (red) indicated
the presence of nanoparticles, while the strong absorptions at 415 (AgNPs) and
413 nm (AgFilm), reveals that nanoparticles give a strong surface plasmon band
in the visible region. In studies with other polymers containing AgNPs were
observed strong absorption around 420nm [21]. This result is in agreement with
previous experiments and predictions for spherical silver nanoparticles with a
mean particle size below 20 nm [22, 23]. The particle size could also to be related
with the intensity and full width half maximum (FWHM) of the plasmon band [22,
23]. It can be observed in Figure 4.2 that FWHM values for nanoparticles
supported in the films (162 nm) were higher than in suspension (83 nm). This
increase in the FWHM can be related with the particle growth [22, 23]. The
104
increase of the nanoparticle happened due to the film production: thermal
treatment (80 °C/1h) of the AgNPs and the drying process. In addition, it is
important to note that the polymeric chains of agar film may have attenuated the
signal absorption, leading to an incoherent interpretation. Thus, other
characterizations were made in this study to clarify this result.
Figure 4.2 – UV-Vis spectroscopy of AgNP and AgFilm.
Source: author.
In Figure 4.3 it is shown the particle size distribution of the AgNPs in the
suspension analyzed by DLS. It was found the value of 76.39 ± 41.86 (mean ±
SD), with a polydispersity index (PDI) of 0.180 which shows AgNP monodisperse.
In a study with nano-bioconjugates was found a single population of AgNP as
revealed by DLS analysis in the size range of 20–40 nm [24]. DLS reading was
also taken to corroborate with the spectroscopic and microscopic findings [25].
Actually, this value represents the hydrodynamic diameter of the particle and is
calculated assuming isotropic spherical particle.
105
Figure 4.3 – DLS.
Source: author.
The FT-IR of AgNPs and AgFilm can be observed in Figure 4.4. The
intensity of the stretching and deformation associated with the methyl groups are
shown in 2,918 cm-1 and 2,851 cm-1 in AgNPs and AgFilm. The bands in the
AgNPs suspension are due to the groups (-C-H) from SDS, used in the production
of these particles, in order to stabilize them. The SDS has a long saturated and
unbranched carbon chain, which has 14 carbons in their structure. Bands are
intensified too in the film because of the groups (-C-H) of the polymer matrix
(agar), that add up to the SDS and increase the presence of groups (-C-H) [26].
The bands observed at 1,215 cm-1 and 1,017 cm-1 are associated with symmetric
and asymmetric stretch of sulfonated groups (-SO4) SDS, respectively [20].
Bands at 3,387 cm-1 from the AgFilm and 3,312 cm-1 of AgNPs can be attributed
to the hydroxyl group stretching vibrations [22]. We observed that in AgNPs they
are more intense, which could be explained by incomplete drying. In addition, we
can see that there was a change in the intensities of the bands of the film and
AgNPs. This is a suggestive factor that occurred chemical bond between the agar
matrix polymer and AgNPs [9, 16]
106
Figure 4.4 – FTIR spectroscopy.
Source: author.
In Figure 4.5 the diffraction angles (2θ) are shown in silver, 38.2°, 44.4°,
64.6° and 77.5° which correspond to planes (111), (200), (220) and (311),
respectively. These diffraction peaks show that AgNPs in AgFilm presented
structure face-centered cubic (fcc) (JCPDS 65-2871) and occurs in nature in
crystalline form and this finding was seen in AgNPs. It is possible to observe the
Bragg reflections (200), (220) and (311) are weaker and wider than the reflection
(111). This finding indicates that the crystal showed growth in a preferential
direction (111), which is common in polymeric films [21, 27]. It was observed in
AgFilm a baseline with several unidentified peaks that are due to the amorphous
material present in the agar mainly galactose residues, which are the constituent
units of the agar [13].
107
Figure 4.5 – XRD.
Source: author.
108
The morphology of the AgNPs in polymer matrix was determined using
SEM and AFM. Figures 4.6a, 4.7a and 4.7b show that these particles had
spherical form and distributed throughout the surface of AgFilm. Figure 4.7b
shows a three-dimensional aspect of the film (topographic mode), confirming the
presence of different phases and characterizing the AgNPs dispersed in the
polymer matrix of the AgFilm [28]. In Figure 4.6 it can be observed EDX spectrum
of the AgFilm in which the silver is about 3.0 keV, as described in the literature
[29]. The EDX confirms the existence of AgNPs in the AgFilm [4]. The
composition of the AgFilm showed various constituents with carbon as main
component, and showed a silver content of 39.49%. The other constituents are
oxygen, sulfur, and sodium chloride all in smaller amount. The origin of these
elements are due to materials used in the production of AgNPs and AgFilms.
Sulfur (S) is derived of the SDS and agarose which is a sulfated polymer, Na and
Cl are present in the reagents.
Histograms size distribution of particles for the AgFilm were constructed
starting from the SEM and AFM images. SEM showed particles with sizes of
87.09 nm ± 28.11 (mean ± SD) as shown at Figure 4.6c and AFM images showed
particles with size of 78.18 nm ± 30.65 (mean ± SD) as shown at Figure 4.7c. The
sizes of AgNPs found in the AgFilm are in agreement when compared by different
techniques: DLS, AFM and SEM. In the SEM and AFM images, we can see
agglomerates formed during the drying process of the AgFilm. These clusters
could explain the high standard deviations found in the histograms of AgNPs –
Figures 4.6c and 4.7c. These clusters are important for the antifungal action, for
extending release of the polymeric matrix AgNPs [30].
109
Figure 4.6 – SEM of AgFilm (a), EDX of AgFilm (b) and histogram for AgNP base on SEM (c).
(a)
(b)
(c)
Source: author.
110
Figure 4.7 – AgFilm AFM (a), AgFilm AFM on topographic mode (b) and histogram for AgNP based on AFM.
(a)
(b)
(c)
Source: author.
111
The antifungal activity of the AgFilm was assessed and the results are
shown in Figure 4.8. Twenty samples of C. albicans were tested and we found
that there was formation of a zone of inhibition of 19.1mm ± 1.8mm (Mean ± SD)
– as can be seen at Figure 4.8c. The antibacterial and antifungal activity of silver-
containing films has been demonstrated in previous work [28]. However, the
number of strains used usually comes down to a strain in our study. It was used
20 C. albicans and obtained excellent results [31]. The antifungal activity of
AgNPs against C. albicans may be due to inhibition of budding process carrying
the destruction of the integrity of the plasma membrane, which would cause the
death of the fungus. We can observe a zone around the disk in which the film had
no fungal growth in Fig. 4.8a and 4.8b. Gels with AgNPs are useful against fungi
and bacteria, and can be an aid to combat these microorganisms [13]. C. albicans
is an opportunistic fungal responsible for many types of skin lesions and agar film
with active AgNPs can be used in bandages to prevent and aid in the treatment
of these infections in burned patients and diabetics who are very vulnerable to
action of the bacteria and fungi [32]. Our results showed high antifungal activity
of films prepared and can be of great use in the food and pharmaceutical industry.
112
Figure 4.8 – Halos around AgFilm (a) and (b) and halo measurement average (c).
(a)
(b)
(c)
Source: author.
5 CONCLUSION
The AgFilm was able to retain the AgNPs in its structure. Also, it showed
good thickness, transparency and resistance. Preliminary results of antifungal
action of nanostructured films with AgNPs indicate that they can be an invaluable
ally in the treatment of skin infections caused by C. albicans, and can be used as
dressing and covers in wound care.
113
REFERENCES
1. Maneerung, T., S. Tokura, and R. Rujiravanit, Impregnation of silver
nanoparticles into bacterial cellulose for antimicrobial wound dressing.
Carbohydrate Polymers, 2008. 72(1): p. 43-51.
2. Rhim, J.-W., Preparation and characterization of bio-nanocomposite
films of agar and silver nanoparticles: Laser ablation method.
Carbohydrate Polymers, 2014. 103: p. 456-465.
3. Cheviron, P., F. Gouanvé, and E. Espuche, Green synthesis of colloid
silver nanoparticles and resulting biodegradable starch/silver
nanocomposites. Carbohydrate Polymers, 2014. 108: p. 291-298.
4. Hassabo, A.G., Impregnation of silver nanoparticles into polysaccharide
substrates and their properties. Carbohydrate Polymers, 2015. 122: p.
343-350.
5. Fortunati, E., Nano-biocomposite films with modified cellulose
nanocrystals and synthesized silver nanoparticles. Carbohydrate
Polymers, 2014. 101: p. 1122-1133.
6. Wang, Y., Preparation of silver nanoparticles dispersed in
polyacrylonitrile nanofiber film spun by electrospinning. Materials Letters,
2005. 59(24–25): p. 3046-3049.
7. Rhim, J.-W., H.-M. Park, and C.-S. Ha, Bio-nanocomposites for food
packaging applications. Progress in Polymer Science, 2013. 38(10–11):
p. 1629-1652.
8. Seo, S.Y., Alginate-based composite sponge containing silver
nanoparticles synthesized in situ. Carbohydrate Polymers, 2012. 90(1):
p. 109-115.
9. Ghosh, S., Antimicrobial activity of highly stable silver nanoparticles
embedded in agar–agar matrix as a thin film. Carbohydrate Research,
2010. 345(15): p. 2220-2227.
114
10. Muthuswamy, E., Highly stable Ag nanoparticles in agar-agar matrix as
inorganic–organic hybrid. Journal of Nanoparticle Research, 2007. 9(4):
p. 561-567.
11. Silva, T.H., Materials of marine origin: a review on polymers and
ceramics of biomedical interest. International Materials Reviews, 2012.
57(5): p. 276-306.
12. Rhim, J.W., L.F. Wang, and S.I. Hong, Preparation and characterization
of agar/silver nanoparticles composite films with antimicrobial activity.
Food Hydrocolloids, 2013. 33(2): p. 327-335.
13. Shukla, M.K., Synthesis and characterization of agar-based silver
nanoparticles and nanocomposite film with antibacterial applications.
Bioresource Technology, 2012. 107: p. 295-300.
14. Ramos, L.d.S., Protease and phospholipase activities of Candida spp.
isolated from cutaneous candidiasis. Revista Iberoamericana de
Micología, 2015. 32(2): p. 122-125.
15. Gajbhiye, M., Fungus-mediated synthesis of silver nanoparticles and
their activity against pathogenic fungi in combination with fluconazole.
Nanomedicine: Nanotechnology, Biology and Medicine. 5(4): p. 382-386.
16. Kanmani, P. and J.-W. Rhim, Properties and characterization of
bionanocomposite films prepared with various biopolymers and ZnO
nanoparticles. Carbohydrate Polymers, 2014. 106: p. 190-199.
17. Kahrilas, G.A., Investigation of Antibacterial Activity by Silver
Nanoparticles Prepared by Microwave-Assisted Green Syntheses with
Soluble Starch, Dextrose, and Arabinose. ACS Sustainable Chemistry &
Engineering, 2014. 2(4): p. 590-598.
18. Pettegrew, C., Silver Nanoparticle Synthesis Using Monosaccharides
and Their Growth Inhibitory Activity against Gram-Negative and Positive
Bacteria. ISRN Nanotechnology, 2014. 2014: p. 8.
115
19. Tan, Z., Oriented growth behavior of Ag nanoparticles using SDS as a
shape director. Journal of Colloid and Interface Science, 2010. 348(1): p.
289-292.
20. Azócar, I., Preparation and antibacterial properties of hybrid-zirconia
films with silver nanoparticles. Materials Chemistry and Physics, 2012.
137(1): p. 396-403.
21. Zhao, X., Microwave-assisted synthesis of silver nanoparticles using
sodium alginate and their antibacterial activity. Colloids and Surfaces A:
Physicochemical and Engineering Aspects, 2014. 444: p. 180-188.
22. Slistan-Grijalva, A., Assessment of growth of silver nanoparticles
synthesized from an ethylene glycol–silver nitrate–polyvinylpyrrolidone
solution. Physica E: Low-dimensional Systems and Nanostructures,
2005. 25(4): p. 438-448.
23. Slistan-Grijalva, A., Classical theoretical characterization of the surface
plasmon absorption band for silver spherical nanoparticles suspended in
water and ethylene glycol. Physica E: Low-dimensional Systems and
Nanostructures, 2005. 27(1–2): p. 104-112.
24. Goswami, A.M., T.S. Sarkar, and S. Ghosh, An Ecofriendly synthesis of
silver nano-bioconjugates by Penicillium citrinum (MTCC9999) and its
antimicrobial effect. AMB Express, 2013. 3: p. 16-16.
25. Ravindran, A., Selective colorimetric sensing of cysteine in aqueous
solutions using silver nanoparticles in the presence of Cr3+. Talanta,
2011. 85(1): p. 533-540.
26. Viana, R.B., A.B.F. da Silva, and A.S. Pimentel, Infrared Spectroscopy of
Anionic, Cationic, and Zwitterionic Surfactants. Advances in Physical
Chemistry, 2012. 2012: p. 14.
27. Pandey, S., G.K. Goswami, and K.K. Nanda, Green synthesis of
biopolymer–silver nanoparticle nanocomposite: An optical sensor for
ammonia detection. International Journal of Biological Macromolecules,
2012. 51(4): p. 583-589.
116
28. Pinto, R.J.B., Antibacterial activity of optically transparent
nanocomposite films based on chitosan or its derivatives and silver
nanoparticles. Carbohydrate Research, 2012. 348: p. 77-83.
29. Kim, S.-C., Antifungal effects of 3D scaffold type gelatin/Ag
nanoparticles biocomposite prepared by solution plasma processing.
Current Applied Physics, 2013. 13, Supplement 1: p. S48-S53.
30. Tran, P.A., D.M. Hocking, and A.J. O'Connor, In situ formation of
antimicrobial silver nanoparticles and the impregnation of hydrophobic
polycaprolactone matrix for antimicrobial medical device applications.
Materials Science and Engineering: C, 2015. 47: p. 63-69.
31. Pinto, R.J.B., Antifungal activity of transparent nanocomposite thin films
of pullulan and silver against Aspergillus niger. Colloids and Surfaces B:
Biointerfaces, 2013. 103: p. 143-148.
32. Peršin, Z., Novel cellulose based materials for safe and efficient wound
treatment. Carbohydrate Polymers, 2014. 100: p. 55-64.
117
CHAPTER 5
GREEN SYNTHESIZED SILVER NANOPARTICLES:
DEVELOPMENT OF GEL, ANTIBACTERIAL ACTIVITY
AND STABILITY STUDIES
118
ABSTRACT
This study was performed to verify the stability, physicochemical properties and
antibacterial activity of a gel functionalized with silver nanoparticles (AgNPs)
obtained by green synthesis. Three formulations of Gel-AgNP were made: 0.5,
1.0 and 2.0% of AgNPs. The gel is skin-compatible and had shown interesting
rheological properties, assuming a pseudoplastic profile. In addition, the stability
of the gel was obtained and it was extremely satisfactory for six months. The
antibacterial activity was performed against various Gram-positive and Gram-
negative bacterias (Escherichia coli, Pseudomonas aeruginosa and
Staphylococcus aureus), showing satisfactory results for 1.0 and 2.0% AgNPs
gels trough well-diffusion technique. The AgNPs gel could provide an alternative
to conventional antibacterial formulations (topical use) for veterinary or human
applications.
Keywords: Silver nanoparticles; Carbomer gel; Green synthesis; Antibacterial
activity
119
CHAPTER 5 – GREEN SYNTHESIZED SILVER NANOPARTICLES:
DEVELOPMENT OF GEL, ANTIBACTERIAL ACTIVITY AND STABILITY
STUDIES
1 INTRODUCTION
Due to the emergency and the raising of multi-resistant microorganisms,
many researches have been performed to achieve a viable alternative in
treatment of patients infected by those pathogens. In this sense, silver
nanoparticles – AgNPs – has been used as paint with antibacterial properties [1],
wound dressing, also with anti-inflammatory and antimicrobial properties [2-4],
cosmetic industries [5] and large use as growth controlling antimicrobial [6-11].
Once the new antibiotics demand a lot of time and money to be developed, and
still there is the risk of the inefficacy of them, even if they are newly released,
because the microorganism may previously develop resistance to them [12, 13].
Antiseptics silver-based have been largely employed due to its large spectrum of
activity against microorganisms and low resistance-inducing of them, when
compared to antibiotics [14].
The activity of silver salts against bacteria has been reported since the
old ages [15] and the silver is used, nowadays, to control the growth of bacteria
in a large scale of applications, such as dental works, catheters and burns [16,
17]. Silver ions and compounds containing silver are, actually, extremely toxic to
microorganisms, showing biocides effects in lot of bacteria [18]. Aymonier and
co-workers [19] published a work showing functionalized AgNPs with amphiphilic
macromolecules associated to antimicrobials as capping agents. AgNPs show
more intensive activity against microorganisms than their ions and salts of silver
[20-23]. Beyond the activity against bacteria and viruses [18, 22, 24, 25], these
nanoparticles had been used also to treat immunologic and inflammatory
disorders [26]. They are also targets of researches due to its unique
physicochemical properties, such as catalytic activities, optic and electronic
properties, antimicrobial activity and magnetic properties [27-29]. Among metallic
nanoparticles, the silver are the ones that show the best bacteriostatic and
bactericide effects [28]. Due to these reasons, the research concerning AgNPs
had increased on the last years [30, 31].
120
According the resistance of microorganism to currently available
antibiotics, AgNPs show themselves as a new and effective alternative to combat
pathogens, once its use does not induce the resistance, even its mechanisms are
not elucidated yet. A viable alternative would be using silver as synergic agent to
the antibiotics, however, this alternative needs more studies and data [32, 33].
Here, we discuss the synthesis and characterization of AgNPs stabilized
colloidal suspension to be used in a gel formulation for topical application. We
also investigated the gel stability up to 6 months and the antibacterial activity.
Jadhav and coworkers [34] produced a functionalized gel with AgNPs to treat
infections in burns. These nanoparticles were obtained by green synthesis with
an aqueous extract of Ammania baccifera. Similar to our work, this author used
carbomer 934 as gelling agent. This product was very effective against
Pseudomonas aeruginosa and Staphylococcus aureus. Another work [35] reports
AgNPs as antimicrobial agent into a gel matrix for topical use. The antimicrobial
activity was observed against fungi and bacteria, and satisfactory results were
obtained. Also, Pérez and coworkers developed a chitosan gel functionalized with
AgNPs and verified the cytotoxicity over human fibroblasts, showing a significant
biocompatibility on their research [36].
2 EXPERIMENTAL
2.1 Materials
The chemical reagents for this study were carbomer 940 (Quimica, São
Paulo, Brazil), sodium carboxymethylcellulose (CMC) (Quimica, São Paulo,
Brazil), silver sulfadiazine (Silvestre Laboratory, Rio de Janeiro, Brazil),
Trietanolamine (BASF, São Paulo, Brazil), silver nitrate from (Dinâmica, São
Paulo, Brazil), ribose (Sigma-Aldrich, São Paulo, Brazil), sodium citrate (Neon,
São Paulo, Brazil) and propylene glycol (Mapric, São Paulo, Brazil). All chemicals
were used without further purification.
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2.2 Methods
2.2.1 Synthesis and functionalization of AgNPs
AgNPs were synthetized starting from AgNO3 0.1mM solutions. The
reduction of Ag+ ions was performed by 1.0g of ribose, and 0.5g of sodium citrate
were used as stabilizer agent, see step in Figure 5.1a. The temperatures of
synthesis were 60, 80 and 100 ºC during 10 minutes in each sample (AgRC-01,
AgRC-02 and AgRC-03, respectively). Then, after the procedure of synthesis, the
solution was centrifuged by 20 minutes at 10000rpm, as shown at Figure 5.1b
and 5.1c. The centrifuged fluid was then transferred to a vial – Figure 5.1d – and
stored at a freezer, 5ºC untill its use. The gels – Figure 5.1e – were made with
AgNPs using 0.5, 1.0 and 2.0% w/w.
Figure 5.1 – Obtaining the gel functionalized with silver nanoparticles.
Source: author.
122
2.2.2 AgNPs characterization
AgNPs were identified by UV-VIS spectroscopy through a GENESYS
10S UV-Vis spectrophotometer. The absorption band near 400nm is a
characteristic pattern of AgNPs.
A Rigaku (Tokyo, Japan) X-ray powder diffractometer operating on 40
kV/30 mA with a Cu-KI tube ( = 1.54056 Å) was used to obtain the X-ray
diffraction pattern of the AgNPs. The diffraction patterns were carried out using
Bragg-Brentano geometry in continuous mode with speed of 1°/min and step size
of 0.02° (2) in the angular range 20–80° (2). 1.0mL of the AgNPs suspensions
was dried at plates of glass under a temperature of 60 ºC and the measure was
done on the film surface.
The scanning electron micrography (SEM) was performed at an
INSPECT 50 SEM with EDS/EBSD and lithography.
2.2.3 Formulation of AgNPs based gel.
Definite amount of CMC and carbomer 940 were dissolved in deionized
water, mixed using Omni Mixer Homogenizer (Model M50) and agitated for 30
min. Propylene glycol was added to the mixture and agitated for additional 10
min. The dispersion was then allowed to hydrate and swell for 60 min. The pH of
the neutralized sample was then measured. The carbopol dispersion was then
neutralized with 98% trietanolamine until the desired pH value of 6.0. During
neutralization, the mixture was stirred gently with a spatula until homogeneous
gel was formed. AgNPs from AgRC-03 were selected to add in three different
concentrations (0.5, 1.0 and 2.0% w/w). These gel formulations were packaged
under sterile conditions, labeled with appropriate details and stored at room
temperature for further use. This last procedure were illustrated at Figure 5.1e
and 5.1f.
2.2.4 Antibacterial activity
Gel silver nanoparticles (Gel-AgNP) in three different concentrations 0.5,
1.0 and 2.0% w/w were tested for antibacterial activity as method suggested by
Srinivasulu et al. [37] using various Gram-positive and Gram-negative bacteria
123
(Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) by the agar
well-diffusion method. Approximately, 20 mL of nutrient agar medium was poured
into sterilized petri-dishes. The bacterial test organisms were grown in nutrient
broth for 24 h. A 100 μL nutrient broth culture of each bacterial organism (1 × 105
CFU ml−1) was used to prepare bacterial lawns. Agar wells of 8 mm diameter
were prepared with the sterilized stainless steel cork borer. The wells were loaded
with 60 μL of Gel-AgNP, 60 μL of culture with gel without AgNPs as a negative
control, along with 60 μL of 1.0% silver sulfadiazine as a positive control. The
plates were incubated at 37 °C for 24 h and then were examined for the presence
of zones of inhibition.
2.2.5 Stability evaluation
The experimental protocol was based on the ICH guideline Stability
Testing of new Drug Substances and Products [38]. Three batches of the
formulation were produced under similar conditions and were then stored at room
temperature (real-time, 25 ± 2°C / 60RH ± 5% humidity) for 6 months and
submitted to accelerated aging for 90 days (oven at 40±2°C / 75RH ± 5%
humidity). The stability of the formulation was defined by samples analysis
without significant changes in parameters physicochemical. The stability samples
(n=3) were taken for analysis at the end of the following time periods: 30, 60, 90
and 180 days.
The apparent viscosity and rheological profile were evaluated using a
Brookfield R/S –CC+ rotational viscometer ® equipped with V3 40/20 spindle.
The pH was controlled using a potentiometric method (pH meter Metrohm ® pH
Meter 744, glass electrode).
124
3 RESULTS AND DISCUSSION
3.1 AgNPs characterization
The AgNPs used in this work were obtained from non-toxic reactants,
which endorses its use as a component in skin-compatible gels, once only ribose
(sugar) and sodium citrate (capping agent in water) are used in this synthesis.
UV-Vis spectroscopy had shown as an excellent technique to
characterize AgNPs. The characteristic band with max= 420nm is an indicative of
its formation and is called plasmon band, which works as a fingerprint to this type
of product [39-42]. The absorption bands of the three different methods of
synthesis of AgNPs are shown in Figure 5.2.
Figure 5.2 – UV–VIS absorption spectra of AgNPs.
Source: author.
It can be observed that the raising of temperature contributes to more
monodisperses nanoparticles, according to the profile of the spectrum.
Figure 5.3 shows the XRPD patterns of the AgNPs samples (AgRC-01,
AgRC-02 and AgRC-03). This technique complements the UV-Vis spectroscopy,
125
showing the silver phases. As verified in other works [42-45], the diffractogram is
characteristic of metallic silver (JCPDS-04-0783) with cubic phase and space
group Fm3m.
Figure 5.3 – XRPD patterns of the AgNPs and diffraction peaks from JCPDS (04-
0783) used for identification and comparison.
Source: author.
AgNPs samples were dried and weighted. Then, in each one of the plates
were droped 1.0mL of silver nanoparticles solution and then warmed up to 100ºC
to evaporate the solvent. The residual on the plates were then wighed and each
sample had shown the following values: 6.7mg (AgRC-01); 6.6mg (AgRC-02) and
6.7mg (AgRC-03). This empiric method gives a reasonable estimative of how
much silver exists in each sample. After this determination, the solution were then
diluted to 4.0 g/L solutions to be used at antimicrobial tests.
Figure 5.4a and 5.4b show the micrographs of the sample AgRC-3 at an
amplification of 100,000X (a) and 238,000X (b), respectively. As it can be seen,
AgNPs show a spheric morphology as predominant geometry, even that others
types of particles are present, such as prismatic and as rod particles – Figure
126
5.4b. Finally, the histogram is shown at Figure 5.4c. The distrubution of AgNPs
shows the frequency and range of different sizes of the particles obtained by the
synthesis. This results corroborates the literature [46-50] that shows the
predominance of spheric silver nanoparticles when the plasmon bands are
assigned to the region of 400 nm.
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).
(a)
(b)
(c)
Source: author.
3.2 AgNPs based gel and properties
Figures 5.5a, 5.5b and 5.5c present the results of rheological analyses
for formulated gel samples.
127
Figure 5.5 – Shear stress (a), viscosity x deformation (b) and viscosity x time
(c).
(a)
(b)
128
(c)
Source: author.
Gel-AgNP 0.5%, Gel-AgNP 1% and Gel-AgNP 2% had shown apparently
mean viscosity as 1.8357Pas, 3.6840Pas and 3.3636Pas, respectively. These
values may be influenced by morphology, concentration and interaction of the
AgNPs with the gel. Due to its high superficial area, AgNPs show a high tendency
to aggregate to minimize the total energy of system, which involves both attractive
and repulsive forces [51]. The surface charges may offer physical stability to the
system, avoiding the agglomeration of nanoparticles through electrostatic
repulsion [52]. These charges had been also explored to improve the interaction
between nanoparticles and skin [53, 54]. According to the obtained data, these
forces cause the changing at viscosity of the gel samples. Here, all the samples
possess a pseudoplastic profile. Pseudoplastic materials have the apparent
viscosity decreased according as the deformation rate increases. So, it cannot
be expressed through anisolated number [55]. The deformation rate is also
function of shear (shown in Figure 5(a)) [56]. It was verified that all gels showed
a distinction between the ascendant and descendant curves. This effect shows
that the fluid is independent of the time and the hysteresis between the curves is
an indicative of thixotropic behavior [57]. This profile is high viable to the gels,
because they deform during application, what makes easy the spreading, but the
129
viscosity returns to its original value when the process is over. Then, the product
cannot drain [58].
The physicochemical properties for formulated gel with 1.0% (Gel-
AgNP1.0%) and 2.0% (Gel-AgNP2.0%) are presented in the Tables 5.1 and 5.2,
with the variation of pH along 2 and 6 months, respectively, when the gels were
stored at 25ºC/60%RH and 40ºC/75%RH. The produced gel was transparent,
light brown, uniform in appearance and without smell after fabrication. The pH
value was 5.8 ± 0.05 (n=6), which is a physiologically acceptable. The 0.5% Gel-
AgNP did not show antimicrobial properties, so its further physicochemical
analysis was not performed.
Table 5.1 – Physicochemical characteristics of 1.0% and 2.0% Gel-AgNP stored
at 25ºC /60%RH.
Gel-AgNP 25ºC /60%RH
Months Color Smell/Odor Appearance pH a,b
1% 2% 1% 2% 1% 2% 1% 2% 0 NC NC NC NC NC NC 5.8±0.4 5.8±0.6 1 NC NC NC NC NC NC 5.9±0.25 5.8±0.4 2 NC NC NC NC NC NC 5.7±0.1 5.8±0.4 3 NC NC NC NC NC NC 5.8±0.08 6.1±0.5 6 NC NC NC NC NC NC 6.0±0.3 6.1±0.8
a= Mean ± SD, n =3, b= statistically significant difference vs. time zero= p = 0.003 NC= Not change.
Table 5.2 –Physicochemical characteristics of 1.0% and 2.0% AgNP gel stored
at 40ºC /75%RH.
Gel-AgNP 40ºC/75% RH
Months Color Smell/Odor Appearance pH a,b
0 NC NC NC 5.8±0.4 1 Dark Brown NC PPT 6.4±1.5 2 Dark Brown NC PPT 7.2±1.8 3 Dark Brown NC PPT 7.6 ± 0.8
a= Mean ± SD, n =3, b= statistically significant difference vs. time zero, p = 0.003.
The stability evaluation of gel containing AgNPs (1.0% and 2.0%)
subjected to accelerated stability conditions exhibited excellent stability with
respect to AgNPs concentration, except at 40ºC/75% relative humidity (RH), as
shown in Table 5.3.
130
Table 5.3 – Chemical stability of AgNP gel (1.0 and 2.0%) during stability testing AgNPs content (%)a.
Gel-AgNP 1% Gel-AgNP 2%
Months 25ºC /60%RH 40ºC/75% RH 25ºC /60%RH 40ºC/75% RH
0 100.0% 100.0% 100.0% 100.0% 1 99.8±0,2 96 ±0,8 99.2±0.08 93 ±1,5 2 99.5±0,5 92.1± 1.5 98.7±0.2 90.1± 1.1 3 98.9±0.1 87.2± 1.2 97.5±0.1 82.7± 0.5b 4 98.1±0.2 97.1±0.8 5 98.9±1.5 96.5±1.6 6 98.6±0,7 95.6±0.5
a= Mean ± SD, n =3, b= statistically significant difference vs. time zero= p = 0.003.
The AgNPs assay values for the 1.0% AgNP gel sample stored at 25ºC
/60%RH were found to range between 100.0 and 98.6 % after 1, 2, 3, 4, 5 and 6
months of storage. This results indicating that the 1.0% AgNP gel is stable at
25%/60RH. However, the assay values for the Gel-AgNP samples stored at
40ºC/75% RH were 96 ±0,8, 92.1± 1.5 and 87.2± 1.5 after 1,2 and 3 moths,
respectively, indicating loss of SN after three months, this mean that after three
months of stability study at 40ºC/75% RH, the Gel-AgNP 1% is not stable. For
Gel-AgNP 2% sample stored at 25ºC /60%RH was found to range between 100.0
and 95.6% after 1, 2, 3, 4, 5 and 6 months of storage and compared to the initial
value 100.0% the observed differences in assay values not statistically
significant, indicating that the Gel-AgNP 2% is stable at 25%/60RH. However,
these values for the gel samples stored at 40ºC/75% RH were 93 ±1,5, 90.1±
1and 82.7± 0.5 after 1, 2 and 3 moths, respectively, indicating loss of AgNPs after
three months. The results are plotted at Figure 5.6a and 5.6b, respectively.
131
Figure 5.6 – Chemical stability of AgNP during the stability testing of gel (1 and
2%) at 25°C (a) and 40°C (b).
(a)
(b)
Source: author.
132
Silver has been used since time immemorial in the form of metallic silver,
silver nitrate, silver sulfadiazine for the treatment of burns, wounds and several
bacterial infections. However, due to the emergence of several antibiotics the use
of these silver compounds has been declined remarkably. Silver in the form of
AgNPs has made a remarkable comeback as a potential antimicrobial agent and
proved to be most effective as it has good antimicrobial efficacy against bacteria,
viruses and other eukaryotic micro-organisms [21]. The use of silver
nanoparticles is also important, as several pathogenic bacteria have developed
resistance against various antibiotics. For this reason, the antibacterial activity of
Gel-AgNP was investigated against various pathogenic Gram-positive and Gram-
negative bacteria, like Staphylococcus aureus (Figure 5.7a), Escherichia coli
(Figure 5.7b) and Pseudomonas aeruginosa (Figure 5.7c) and. The highest
antimicrobial activity was observed against, E. coli (negative), P. aeruginosa and
S. aureus for gel samples with 1 and 2% concentrations of AgNPs. Not found
antimicrobial activity for the gel 0,5% of silver nanoparticles to E. coli and P.
aeruginosa. The Gram negative bacteria E. coli and P. aeruginosa were less
sensitive to Gel - AgNp with 0,5% compared with S. aureus. This result was
similar to those found by Sondi and Salopek-Sondi [59] This can be explain due
to the characteristics of certain bacterial species. The tests are shown in Figures
5.7a and 5.7b. Feng et al. [60] reported the mechanism of AgNPs action and used
E. coli and S. aureus as model organisms. The nanoparticles get attached to the
cell membrane and also penetrate inside the bacteria. The bacterial membrane
contains sulfur-containing proteins and the AgNPs interact with these proteins in
the cell as well as with the phosphorus-containing compounds like DNA.
Nanoparticles preferably attack the respiratory chain, cell division finally leading
to cell death. Silver nanoparticles had emerged up with diverse medical
applications in silver-based dressings [61].
133
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).
(a)
(b)
134
(c)
Source: author.
4 CONCLUSION
The gel functionalized with silver nanoparticles (1.0 and 2.0%) had shown
effective action against the strains of related bacteria on this work. These results
sign for a further study, investigating its activity at higher concentrations, for
example. The physicochemical properties had shown the skin compatibility (pH)
of the formulations, nicer odor and color, such as the rheological behavior is
satisfactory. About the stability, the formulations had shown a good profile for
25ºC/60ºRH for six months.
135
REFERENCES
1. Ataeefard, M. and S. Sharifi, Antibacterial flexographic ink containing silver nanoparticles. Progress in Organic Coatings, 2014. 77(1): p. 118-123.
2. Said, J., An in vitro test of the efficacy of silver-containing wound dressings against Staphylococcus aureus and Pseudomonas aeruginosa in simulated wound fluid. International Journal of Pharmaceutics, 2014. 462(1–2): p. 123-128.
3. Gaisford, S., An in vitro method for the quantitative determination of the antimicrobial efficacy of silver-containing wound dressings. International Journal of Pharmaceutics, 2009. 366(1–2): p. 111-116.
4. Hebeish, A., Antimicrobial wound dressing and anti-inflammatory efficacy of silver nanoparticles. International Journal of Biological Macromolecules, 2014. 65: p. 509-515.
5. Sallum, L.F., Optimization of SERS scattering by Ag-NPs-coated filter paper for quantification of nicotinamide in a cosmetic formulation. Talanta, 2014. 118: p. 353-358.
6. Gu, C., H. Zhang, and M. Lang, Preparation of mono-dispersed silver nanoparticles assisted by chitosan-g-poly(ɛ-caprolactone) micelles and their antimicrobial application. Applied Surface Science, 2014. 301: p. 273-279.
7. Mohan, S., Completely green synthesis of dextrose reduced silver nanoparticles, its antimicrobial and sensing properties. Carbohydrate Polymers, 2014. 106: p. 469-474.
8. Lokina, S., Cytotoxicity and antimicrobial activities of green synthesized silver nanoparticles. European Journal of Medicinal Chemistry, 2014. 76: p. 256-263.
9. Rhim, J.W., L.F. Wang, and S.I. Hong, Preparation and characterization of agar/silver nanoparticles composite films with antimicrobial activity. Food Hydrocolloids, 2013. 33(2): p. 327-335.
10. Akhavan, A., Synthesis of antimicrobial silver/hydroxyapatite nanocomposite by gamma irradiation. Radiation Physics and Chemistry, 2014. 98: p. 46-50.
11. Kanmani, P. and J.-W. Rhim, Physicochemical properties of gelatin/silver nanoparticle antimicrobial composite films. Food Chemistry, 2014. 148: p. 162-169.
12. Rai, M., Broad-spectrum bioactivities of silver nanoparticles: the emerging trends and future prospects. Applied Microbiology and Biotechnology, 2014. 98(5): p. 1951-1961.
136
13. Nordmann, P., Carbapenemase-producing Enterobacteriaceae: Overview of a major public health challenge. Médecine et Maladies Infectieuses, 2014. 44(2): p. 51-56.
14. Jones, S.A., Controlling wound bioburden with a novel silver-containing Hydrofiber® dressing. Wound Repair and Regeneration, 2004. 12(3): p. 288-294.
15. Silver, S. and L.T. Phung, Bacterial heavy metal resistance: New Surprises. Annual Review of Microbiology, 1996. 50(1): p. 753-789.
16. M, C., Antibacterial and bioactive silver-containing Na2OCaO2SiO2 glass prepared by sol-gel method. Journal of Materials Science and Materials for Medicine, 2004. 15: p. 7.
17. Crabtree, J., The efficacy of silver-ion implanted catheters in reducing peritoneal dialysis-related infections. Peritoneal Dialysis International, 2003. 23(4): p. 368-374.
18. Zhao, G. and J.S. Stevens, Multiple parameters for the comprehensive evaluation of the susceptibility of Escherichia coli to the silver ion. . Biomaterials, 1998. 11: p. 6.
19. Aymonier, C., Hybrids of silver nanoparticles with amphiphilic hyperbranched macromolecules exhibiting antimicrobial properties. Chemical Communications, 2002(24): p. 3018-3019.
20. Ansari, M., Evaluation of antibacterial activity of silver nanoparticles against MSSA and MRSA on isolates from skin infections. Biology and Medicine, 2011.
21. Libor, K. and S. Jana, Comment on 'Preparation and antibacterial activity of Fe 3 O 4 @Ag nanoparticles'. Nanotechnology, 2009. 20(2): p. 028001.
22. Rai, M., A. Yadav, and A. Gade, Silver nanoparticles as a new generation of antimicrobials. Biotechnology Advances, 2009. 27(1): p. 76-83.
23. Lok, C.-N., Proteomic Analysis of the Mode of Antibacterial Action of Silver Nanoparticles. Journal of Proteome Research, 2006. 5(4): p. 916-924.
24. Kim, J.S., Antimicrobial effects of silver nanoparticles. Nanomedicine: Nanotechnology, Biology and Medicine, 2007. 3(1): p. 95-101.
25. Magaña, S.M., Antibacterial activity of montmorillonites modified with silver. Journal of Molecular Catalysis A: Chemical, 2008. 281(1–2): p. 192-199.
137
26. Shin, S.-H., The effects of nano-silver on the proliferation and cytokine expression by peripheral blood mononuclear cells. International Immunopharmacology, 2007. 7(13): p. 1813-1818.
27. Meenal, K., Extracellular synthesis of silver nanoparticles by a silver-tolerant yeast strain MKY3. Nanotechnology, 2003. 14(1): p. 95.
28. Souza, G.I.H., Utilization of Fusarium oxysporum in the biosynthesis of silver nanoparticles and its antibacterial activities. , in Xth National Meeting of Environmental Microbiology. 2004: Curitiba, PR (Brazil)
29. Durán, N., Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporum strains. Journal of Nanobiotechnology, 2005. 3: p. 8-8.
30. Kim, T.N., Antimicrobial effects of metal ions (Ag+, Cu2+, Zn2+) in hydroxyapatite. Journal of Materials Science: Materials in Medicine, 1998. 9(3): p. 129-134.
31. Cho, K.-H., The study of antimicrobial activity and preservative effects of nanosilver ingredient. Electrochimica Acta, 2005. 51(5): p. 956-960.
32. Wright, G.D., Bacterial resistance to antibiotics: Enzymatic degradation and modification. Advanced Drug Delivery Reviews, 2005. 57(10): p. 1451-1470.
33. Wright, G.D., Resisting resistance: new chemical strategies for battling superbugs. Chemistry & Biology, 2000. 7(6): p. R127-R132.
34. Jadhav, K., Green and ecofriendly synthesis of silver nanoparticles: Characterization, biocompatibility studies and gel formulation for treatment of infections in burns. Journal of Photochemistry and Photobiology B: Biology, 2016. 155: p. 109-115.
35. Jain, J., Silver Nanoparticles in Therapeutics: Development of an Antimicrobial Gel Formulation for Topical Use. Molecular Pharmaceutics, 2009. 6(5): p. 1388-1401.
36. Pérez-Díaz, M., Anti-biofilm activity of chitosan gels formulated with silver nanoparticles and their cytotoxic effect on human fibroblasts. Materials Science and Engineering: C, 2016. 60: p. 317-323.
37. Srinivasulu, B., Neomycin production with free and immobilized cells of Streptomyces marinensis in an airlift reactor. Process Biochemistry, 2002. 38(4): p. 593-598.
38. ICH Topic Q1 A (R2) Stability Testing of new Drug Substances and Products in International Conference on Harmonisation of Technical requirements for Registration of Pharmaceuticals for Human Use. 2005. Geneva, Switzerland.
138
39. Rastegarzadeh, S. and F. Hashemi, A surface plasmon resonance sensing method for determining captopril based on in situ formation of silver nanoparticles using ascorbic acid. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2014. 122: p. 536-541.
40. Liu, P., Fluorescence enhancement of quercetin complexes by silver nanoparticles and its analytical application. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2014. 122: p. 238-245.
41. Chhatre, A., Color and surface plasmon effects in nanoparticle systems: Case of silver nanoparticles prepared by microemulsion route. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2012. 404: p. 83-92.
42. Bindhu, M.R. and M. Umadevi, Surface plasmon resonance optical sensor and antibacterial activities of biosynthesized silver nanoparticles. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2014. 121: p. 596-604.
43. Kumar, D., V. Palanichamy, and S. Roopan, Green synthesis of silver nanoparticles using Alternantheradentata leaf extract at room temperature and their antimicrobial activity. Spectrochim Acta A, 2014. 127: p. 4.
44. Ahluwalia, V., Green synthesis of silver nanoparticles by Trichoderma harzianum and their bio-efficacy evaluation against Staphylococcus aureus and Klebsiella pneumonia. Industrial Crops and Products, 2014. 55: p. 202-206.
45. Gade, A., Green synthesis of silver nanoparticles by Phoma glomerata. Micron, 2014. 59: p. 52-59.
46. Olenin, A.Y., Formation of surface layers on silver nanoparticles in aqueous and water-organic media. Colloid Journal, 2008. 70(1): p. 71-76.
47. Lima, I., Atividade antifúngica de óleos essenciais sobre espécies de Candida. Brazilian Journal of Pharmacognosy, 2006. 6: p. 5.
48. Araujo, J., Ação antimicrobiana de óleos essenciais sobre microrganismos potencialmente causadores de infecções oportunistas. Ver Patologia Tropical, 2004. 33: p. 10.
49. AC, T., Avaliação de atividade bactericida de nanopartículas de prata e ouro impregnadas com antimicrobiano. Criciúma, 2010. 2010, Universidade do Extremo Sul Catarinense,: Criciúma.
50. Arendrup, M., Epidemiology of invasive candidiasis. . Current Opinion in Critical Care, 2010. 6: p. 8.
51. Jiang, J., G. Oberdörster, and P. Biswas, Characterization of size, surface charge, and agglomeration state of nanoparticle dispersions for
139
toxicological studies. Journal of Nanoparticle Research, 2009. 11(1): p. 77-89.
52. Freitas, C. and R.H. Müller, Effect of light and temperature on zeta potential and physical stability in solid lipid nanoparticle (SLN™) dispersions. International Journal of Pharmaceutics, 1998. 168(2): p. 221-229.
53. Peira, E., Positively charged microemulsions for topical application. International Journal of Pharmaceutics, 2008. 346(1–2): p. 119-123.
54. Ridolfi, D.M., Chitosan-solid lipid nanoparticles as carriers for topical delivery of tretinoin. Colloids and Surfaces B: Biointerfaces, 2012. 93: p. 36-40.
55. Martin, A., Physical pharmacy. 4th ed. 1993, Philadelphia: Lea & Febiger.
56. Neves, D. and M. Ogasawara, Correlação comportamento reológico – liberação do princípio ativo de semisólidos, in XVI Congresso Brasileiro de Cosmetologia. 2002: São Paulo - ABC.
57. Tárrega, A., L. Durán, and E. Costell, Flow behaviour of semi-solid dairy desserts. Effect of temperature. International Dairy Journal, 2004. 14(4): p. 345-353.
58. Gaspar, L.R. and P.M.B.G. Maia Campos, Rheological behavior and the SPF of sunscreens. International Journal of Pharmaceutics, 2003. 250(1): p. 35-44.
59. Sondi, I. and B. Salopek-Sondi, Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria. Journal of Colloid and Interface Science, 2004. 275(1): p. 177-182.
60. Feng, Q.L., A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. Journal of Biomedical Material Research, 2000. 52: p. 7.
61. Durán, N., Antibacterial Effect of Silver Nanoparticles Produced by Fungal Process on Textile Fabrics and Their Effluent Treatment. Journal of Biomedical Nanotechnology, 2007. 3(2): p. 203-208.
140
CHAPTER 6
ASSOCIATION BETWEEN SILVER NANOPARTICLES
AND ITRACONAZOLE: PROPOSAL FOR A DELIVERY
SYSTEM OF ANTIFUNGAL.
141
CHAPTER 6 – ASSOCIATION BETWEEN SILVER NANOPARTICLES AND
ITRACONAZOLE: PROPOSAL FOR A DELIVERY SYSTEM OF
ANTIFUNGAL.
1 INTRODUCTION
Infections caused by Candida spp, are causes of death in several
countries and the treatment of these infections is still quite limited, because there
are few drugs available for the treatment [1, 2], and the main species involved
are C. albicans, C. tropicalis and C. parapsilosis [3]. C. parapsilosis is an
emerging specie in Latin America, surpassed only by C. albicans. A high rate of
infection was observed in pediatric patients [4].
Itraconazole (ITR) is an antifungal agent that belongs to the second
generation of azoles, such as triazole, which has broad spectrum, however, has
low aqueous solubility at room temperature [5]. This low solubility limits its use,
and pharmaceutical forms containing ITR are few in number which hinders the
treatment of elderly patients, children, infants and those severely ill [6]. The
challenge is to develop pharmaceutical formulations of ITR without modifying its
antifungal activity [7].
Nanotechnology has the potential to revolutionize medical treatment,
formulating drugs or vehicles more efficient and less toxic, using this new
technological tool. It can solve problems such as low solubility of drugs, low
bioavailability and thus make safer drugs and to even more affordable and
accessible to the most needy populations around the world [8, 9].
Silver nanoparticles (AgNPs) are synthesized in various ways. The
chemical remains being the most known and investigated, among various
applications stand out the antifungal activity [10-12]. AgNPs in solution are
usually unstable and require capping agent to stabilize and one of the most used
is the sodium dodecyl sulfate (SDS). This agent can increase the microbial
activity of AgNPs [13]. SDS is a compound having a long carbon chain (12 carbon
atoms), unsaturated, unbranched and associated with a polar head, this structure
could aid in the solubilization of the ITR and function as a sustained release
system of this antifungal. The objective of this study was to characterize and
evaluate the association of AgNPs and ITR as a delivery system for antifungal
against Candida parapsilosis strains.
142
2 EXPERIMENTAL
2.1 Materials
Silver nitrate (Dinâmica, São Paulo, Brasil), Sodium dodecyl sulfate
(SDS) (Dinâmica, São Paulo, Brasil), itraconazole (Jassen-Cilag, São Paulo,
Brasil), Mueller-Hinton agar (Himedia, India) and Methylene blue (Dinâmica, São
Paulo, Brasil) were used according to the manufacturer’s instructions.
2.2 Methods
2.2.1 Synthesis of AgNPs
AgNPs were produced using glucose as a reducing agent and SDS as
stabilizer. For the production of AgNPs, 1.0g of glucose and 0.5g of SDS were
used. The reagents were weighed and dissolved in 500 mL of 5.0 mM AgNO3
solution. The reaction was accelerated by adding 1.0 mL of 0.2 mol L-1 NaOH.
The mixture was heated to 50°C and maintained at this temperature for 30 min
under constant agitation. When the pale yellow solution appears, it is an indicative
that the reaction was successful. Then, the AgNPs produced were precipitated in
a HIMAC CS150NX ultracentrifuge at 10,000 rpm for 20 min. After each step, the
AgNPs were washed three times with distilled water and stored in refrigerator
until get ready to use.
2.2.2 Association of AgNPs with Itraconazole
The association of AgNPs with ITR was made by following: 50 mL of
centrifuged AgNP solution was mixture with 2.0 mL and 3.0 mL of ITR (80 g mL-
1), respectively. After this dilution, the solution was stirred during 120 minutes
over 200 rpm under room temperature (25 ºC), according to Zhenquan et al
methodology [14]. As control, solutions with AgNP only and ITR only were
submitted to the same procedure and conditions.
143
2.2.3 Antifungal activity of AgNPs + ITR
There were used 50 strains of Candida parapsilosis on this work. These
strains belong to the collection of strains of the Laboratório de Microbiologia de
Leveduras from Departamento de Análises Clínicas e Toxicológicas from
Faculdade de Farmácia of Universidade Federal do Ceará, and were purified and
identified in agar potato medium and chromogenic medium. The antifungal
activity of system was evaluated by diffusion method in Mueller-Hinton medium,
added of glucose 2% and methylene blue 0.05%. In each plate was seeded a
strain, and after this it was made a 6.0 mm diameter orifice on the medium. In
each orifice was added 100 L of the solutions: 1 – Itraconazole 3.2 g mL-1; 2 –
Centrifuged AgNP; 3 – Itraconazole 4.8 g mL-1; 4 – AgNPs + Itraconazole 3.2
g mL-1; 5 – AgNPs + Itraconazole 4.8 g mL-1 and 6 – Itraconazole 80 g mL-1.
The plates were incubated during 24 hours under 35 ºC temperature. After this
period, the inhibition zones around the orifice were measured [10].
2.2.4 Statistic data
The average of zones of antifungal activity of systems with Itraconazole
3.2 g mL-1 and 4.8 g mL-1 were compared to the centrifuged AgNP by using
the t student test, being statistically significant when p<0.05.
2.3 Characterization
2.3.1 UV-Vis spectroscopy
AgNPs absorb light around the 420 nm wavelength, and absorption of
the particles was measured in the UV-Visible range of 300-700 nm in a Thermo
Scientific GENESYS™ 10s spectrophotometer.
144
2.3.2 Infrared spectroscopy
As the surface of the AgNPs shows interaction with the SDS, this
interaction was observed by Fourier Transform Infrared Spectroscopy (FT-IR)
using KBr pellets. These measurements were taken on a Perkin-Elmer FT-IR
Spectrum ONE spectrophotometer, in the range of 4000-500 cm-1 with a
resolution of 4 cm-1.
2.3.3 X-ray diffraction spectroscopy
The pattern of X-ray powder diffraction (XRPD) measurements was
carried out with AgNPs in order to determine the crystallinity thereof. These
samples were oven-dried at 60°C and analysed on a PAN X-Pert Pro MRD
(Amsterdam, Netherlands). The pure sample of AgNPs was placed in a 2.5 cm x
2.5 cm glass support and the diffractogram was measured using Cu-Ka and
nickel radiation with an energy of 40 kV and 30 mA with scanning (2θ) from 30°
to 80° [15].
2.3.4 Dynamic light scattering
Dynamic light scattering (DLS) was used to monitor aggregation and size
of the particles in the Nanozetasizer (Malvern, United States).
2.3.5 Scanning electron microscopy
AgNPs morphology was obtained by Scanning Electron Microscopy
(SEM) using the electron microscope Quanta-450 (FEI) with a field-emission gun
(FEG), a 100 mm stage and an X-ray detector (model 150, Oxford) for energy-
dispersive X-ray spectroscopy (EDX), from Central Analítica – Departamento de
Física from Universidade Federal do Ceará, Campus do Pici. The compositional
map was performed to determine the presence of silver on its constitution (AgNPs
+ ITR).
145
3 RESULTS AND DISCUSSION
Nanotechnology may contribute in a decisive way on treatment of some
human illness. The possibility to utilize these structures as drug carriers may
create new systems of prolonged drug delivery, as well as may be used as
contrast agents, and then reduce the side effects associated to those medicine
[16]. The UV-Vis specter of the AgNPs as well as the system AgNPs + ITR may
be observed at Figure 6.1 below.
Figure 6.1 – UV-Vis spectrum
Source: author.
146
The absorption band of AgNPs is present and shows itself with a
maximum of absorption in 430 nm, which is characteristic of plasmon band for
silver nanoparticles [17]. Other bands are observed with a discreet bathochromic
displacement to 422 nm and 424 nm, respectively to the systems containing
AgNPs and ITR with 3.2 and 4.8 g mL-1. This displacement may be an indicative
of conjugation, according to Kumar and co-workers [18]. The graphic marked with
black refers to ITR, which had shown no absorption.
Also, the UV-Vis technique is really valuable to monitor and investigate
the process of growing of nanoparticles, and is important to know that the
changes on experimental absorption are due to modifications in size or
concentration of particles in solution [19].
The size of the system was evaluated by the dynamic light scattering
(DLS), as can be seen at Figure 6.2a, 6.2b and 6.2c. It is possible to observe that
there was no considerable change into the size of the particles.
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.
(a)
147
(b)
(c)
Source: author.
148
The centrifuged AgNPs had an average size of 88.81 ± 40.64 nm, while
the particles of system AgNPs + ITR 3.2 g mL-1 measured 84.93 ± 81.78 nm
and the last one, AgNPs + ITR 4.8 g mL-1 had shown an average size of 91.99
± 43.81 nm.
To verify the phases of samples, each one was submitted to the X-ray
diffraction (XRD) spectroscopy. It can be seen at Figure 6.3 the diffractogram of
the particles.
Figure 6.3 – XRD of samples (a) centrifuged AgNPs, (b) AgNPs + Itraconazole
3.2 µg mL-1 and (c) AgNPs + Itraconazole 4.8 µg mL-1.
Source: author.
149
The first diffraction pattern shown at (a) refers to the silver nanoparticle
and match exactly with the black bars presented at the bottom of graphic, which
is the Joint Committee on Powder Diffraction Standards (JCPDS) card for silver,
numbered 04-0783. At (b) and (c) it is possible to see the character (*), which
signs a new phase on the mixture. Possible, it is a semi-crystalline phases of the
drug. This halo reveals a crystallo-conformacional appearing at the system, which
occurs when a crystalline phase is mixed with an amorphous or semi-crystalline
one [20].
Another very important parameter is the morphology of system. So, to
investigate this one, the scanning electron microscopy (SEM) was performed.
Concomitant to the SEM, the electron dispersive X-ray (EDX) was also
performed, exactly to identify silver and build the compositional map of the
samples. Also, a histogram was built to compare the sizes obtained by SEM and
the acquired by DLS. The micrographs are shown below at Figures 6.4a, 6.4b
and 6.4c. Following the sequence, the EDX are shown at Figures 6.5a, 6.5b and
6.5c and finally, the built histograms are shown ate Figures 6.6a, 6.6b and 6.6c.
150
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.
(a)
(b)
(c)
Source: author.
151
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.
Source: author.
152
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
(a) (b)
(c)
Source: author.
153
The AgNPs showed spherical shape – what was spected for its UV-Vis
profile [21] – even if not well definite, probably because of the low temperature of
synthesis and the capping agent. When comparing the three acquired
micrographs, it is not possible to identify any remarkable differences among the
samples, which showed prevalence of the spherical particles.
When observing the EDX graphics, it is possible to verify that there was
a high relative concentration of silver on the investigated region of microscopy.
The presences of sulfur and sodium are expected, once the capping agent –
sodium dodecyl sulfate – shows these elements on its constitution. After all, this
technique is a complementary one, which will confirm AgNP in the analyzed
region.
Once the SEM gives the size of nanoparticles (by comparing to the scale
and using Image-J® program), it is possible to build three histograms of the
different systems. When observing the nanoparticles, it is possible to identify
various sizes of nanoparticles and the system containing AgNPs + ITR. As the
SEM is performed into a region of sample, it is normal that the size distribution
be different among them. The sample containing AgNPs only had shown an
average size of 56.2 ± 18.1 nm, while the sample with lower concentration of ITR
had shown 47,5 ± 22,9 nm as average size and the last one, showed the values
66,6 ± 20,08 nm to the average size. It would be expected that the second
investigated sample had higher values than the first, once there were AgNPs and
ITR on its composition. However, as previously said, this technique investigate
restrict regions, and possibly the nanoparticles of the AgNPs sample were higher
than the system AgNPs + ITR of the second one. The last sample showed and
expected result when compared to the first.
Continuing with the investigation, now it is built the compositional map of
samples. This is a complementary technique to elucidate the presence of silver
nanoparticles in the analyzed area of sample during the performing of SEM. The
results are shown ate Figure 6.7.
154
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).
(a)
(c)
(e)
(b)
(d)
(f)
Source: author.
155
So, the compositional map determined the presence of silver around all
the three samples.
The identification of C. parapsilosis was performed trough chromogenic
medium, as can be seen at Figure 6.8 below.
Figure 6.8 – Chromogenic medium to identify C. parapsilosis.
Source: author.
Finally, after the characterization procedures, it was performed the
antifungal test. The results are shown below at Figure 6.9.
156
Figure 6.9 – Sensibility test of C. parapsilosis against six different combinations.
From twelve o’clock and clockwise: ITR 4.8 g mL-1, centrifuged AgNPs, ITR 3.2
g mL-1, ITR 80 g mL-1, AgNPs + ITR 4.8 g mL-1 and ending, AgNPs + ITR 3.2
g mL-1.
Source: author.
The antifungal test showed interesting results. Although both orifices
containing ITR 4.8 g mL-1 and 3.2 g mL-1 had shown no, activity, when
associated to AgNPs, it is possible to see halos. However, there is a halo signaled
to the centrifuged AgNPs, which leads us to deduce that this activity is associated
to the nanoparticles. Even this, the halo of AgNPs + ITR 4.8 g mL-1 is higher
than the ITR control drug and the isolated AgNPs halo. So, it is possible to infer
that at this concentration, the drug shows effect when associated with AgNPs,
and with a considerably lower concentration. This preliminary result may point to
new drugs associations and, with this, reducing the growing of fungi by using
157
lowers concentrations, contributing, also, to avoid of the appearing of resistance
mechanisms on microorganisms. To understand the effect of the association,
Figure 6.10 shows a higher and significant halo of this association.
Figure 6.10 – Itraconazole as control drug and its associations with silver
nanoparticles.
Source: author.
158
To understand these qualitative data, the halos were measured and
compared among them. Figure 6.11 has all the inhibition zones data. It is possible
to see the effectiveness of the system AgNPs + ITR 4.8 mg mL-1 was statistically
significant (p < 0.05) when compared to the centrifuged AgNPs.
Figure 6.11 – Susceptibility testing antifungal agents.
Source: author.
When considering the capping agent – SDS – and its lipophilic chain
surrounding the AgNPs, it is proposed an image to represent the possible
interaction between the itraconazole and silver nanoparticles, which suggests a
structure of controlled delivery system.
159
Figure 6.12 – AgNPs + ITR representative scheme.
Source: author.
4 CONCLUSIONS
The systems investigated had shown that is possible to obtain
satisfactory results when associating AgNPs and itraconazole against C.
parapsilosis, which suggests that itraconazole may be used with lower
concentrations. Also, the sizes of systems were maintained as nanoscaled, which
are the one of the aims of this work.
ACKNOWLEDGEMENTS
The authors gratefully thank the Central Analítica-UFC/CT-INFRA/MCTI-
SISNANO/Pró-Equipamentos, Departamento de Física da UFC, CAPES and
CNPq, for the grant provided to support the research on nanoparticles.
160
REFERENCES
1. Beardsley, J., Estimating the burden of fungal disease in Vietnam.
Mycoses, 2015. 58: p. 101-106.
2. Lagrou, K., Burden of serious fungal infections in Belgium. Mycoses,
2015. 58: p. 1-5.
3. Silva, S., Candida glabrata, Candida parapsilosis and Candida
tropicalis: biology, epidemiology, pathogenicity and antifungal resistance.
FEMS Microbiology Reviews, 2012. 36(2): p. 288-305.
4. Oliveira, V.K.P., FUNGEMIA CAUSED BY Candida SPECIES IN A
CHILDREN'S PUBLIC HOSPITAL IN THE CITY OF SÃO PAULO,
BRAZIL: STUDY IN THE PERIOD 2007-2010. Revista do Instituto de
Medicina Tropical de São Paulo, 2014. 56(4): p. 301-305.
5. Bhardwaj, S.P., Correlation between Molecular Mobility and Physical
Stability of Amorphous Itraconazole. Molecular Pharmaceutics, 2013.
10(2): p. 694-700.
6. Kumar, N., Evaluation of compatibility of itraconazole with excipients
used to develop vesicular colloidal carriers. Journal of Thermal Analysis
and Calorimetry, 2014. 115(3): p. 2415-2422.
7. Cevher, E., Bioadhesive tablets containing cyclodextrin complex of
itraconazole for the treatment of vaginal candidiasis. International Journal
of Biological Macromolecules, 2014. 69: p. 124-136.
8. Gaurav, C., Albumin stabilized silver nanoparticles-clotrimazole [small
beta]-cyclodextrin hybrid nanocomposite for enriched anti-fungal activity
in normal and drug resistant Candida cells. RSC Advances, 2015. 5(87):
p. 71190-71202.
9. Choudhary, S. and V. Kusum Devi, Potential of nanotechnology as a
delivery platform against tuberculosis: Current research review. Journal
of Controlled Release, 2015. 202: p. 65-75.
161
10. MALLMANN, E.J.J., ANTIFUNGAL ACTIVITY OF SILVER
NANOPARTICLES OBTAINED BY GREEN SYNTHESIS. Revista do
Instituto de Medicina Tropical de São Paulo, 2015. 57: p. 165-167.
11. Botequim, D., Nanoparticles and Surfaces Presenting Antifungal,
Antibacterial and Antiviral Properties. Langmuir, 2012. 28(20): p. 7646-
7656.
12. Singh, M., Metallic silver nanoparticle: a therapeutic agent in
combination with antifungal drug against human fungal pathogen.
Bioprocess and Biosystems Engineering, 2013. 36(4): p. 407-415.
13. Kora, A.J., R. Manjusha, and J. Arunachalam, Superior bactericidal
activity of SDS capped silver nanoparticles: Synthesis and
characterization. Materials Science and Engineering: C, 2009. 29(7): p.
2104-2109.
14. Tan, Z., Oriented growth behavior of Ag nanoparticles using SDS as a
shape director. Journal of Colloid and Interface Science, 2010. 348(1): p.
289-292.
15. Pandey, S., G.K. Goswami, and K.K. Nanda, Green synthesis of
biopolymer–silver nanoparticle nanocomposite: An optical sensor for
ammonia detection. International Journal of Biological Macromolecules,
2012. 51(4): p. 583-589.
16. Sapsford, K.E., Functionalizing Nanoparticles with Biological Molecules:
Developing Chemistries that Facilitate Nanotechnology. Chemical
Reviews, 2013. 113(3): p. 1904-2074.
17. Rastegarzadeh, S. and F. Hashemi, A surface plasmon resonance
sensing method for determining captopril based on in situ formation of
silver nanoparticles using ascorbic acid. Spectrochimica Acta Part A:
Molecular and Biomolecular Spectroscopy, 2014. 122: p. 536-541.
18. Kumar, D., V. Palanichamy, and S. Roopan, Green synthesis of silver
nanoparticles using Alternantheradentata leaf extract at room
162
temperature and their antimicrobial activity. Spectrochim Acta A, 2014.
127: p. 4.
19. Bovero, E., Dispersion of Silver Nanoparticles into Polymer Matrix Dry
Adhesives to Achieve Antibacterial Properties, Increased Adhesion, and
Optical Absorption. Macromolecular Reaction Engineering, 2013. 7(11):
p. 624-631.
20. Kayser, Y., J. Sa, and J. Szlachetko, Nanoparticle characterization by
means of scanning free grazing emission X-ray fluorescence. Nanoscale,
2015. 7(20): p. 9320-9330.
21. Panikkanvalappil R. Sajanlal, et al. Anisotropic nanomaterials: structure,
growth, assembly, and functions. 2011 [cited 2016 5/13/2016]; Available
from:
http://www.nanoreviewsexperiments.net/index.php/nano/article/view/588
3/7100.