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PRÓ-REITORIA DE PÓS-GRADUAÇÃO E PESQUISA
ÁREA DE CIÊNCIAS TECNOLÓGICAS
Programa de Pós-Graduação em Nanociências
LEONARDO QUINTANA SOARES LOPES
NANOCÁPSULAS CONTENDO MONOLAURATO DE GLICEROL:
ATIVIDADE ANTIMICROBIANA, ANTIBIOFILME E ASPECTOS
TOXICOLÓGICOS
Santa Maria, RS
2019
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LEONARDO QUINTANA SOARES LOPES
NANOCÁPSULAS CONTENDO MONOLAURATO DE GLICEROL:
ATIVIDADE ANTIMICROBIANA, ANTIBIOFILME E ASPECTOS
TOXICOLÓGICOS
Tese de Doutorado apresentada ao
Programa de Pós-Graduação em
Nanociências da Universidade Franciscana
de Santa Maria, como parte das exigências
para obtenção do título de Doutor em
Nanociências, na área de concentração
Biociências e Nanomateriais.
Orientador(a): Prof. Dr. ROBERTO CHRIST VIANNA SANTOS
Santa Maria, RS
2019
Elaborada pela Bibliotecária Eunice de Olivera CRB 10/1491
L864n Lopes, Leonardo Quintana Soares
Nanocápsulas contendo monolaurato de glicerol:
atividade antimicrobiana, antibiofilme e aspectos
toxicológicos / Leonardo Quintana Soares Lopes ;
orientação Roberto Christ Vianna Santos – Santa Maria :
Universidade Franciscana - UFN, 2019.
124 f. : il.
Tese (Doutorado em Nanociências) Programa de Pós-
Graduação em Nanociências – Universidade Franciscana –
UFN
1. Biofilmes 2. Nanotecnologia 3. Citotoxicidade
4. Rhamdia quelen I. Santos, Roberto Christ Vianna
II. Título
CDU 62
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RESUMO
Os biofilmes são aglomerados microbianos envoltos por uma matriz de polissacarídeos
extracelular. No século 21 ficou comprovado que a capacidade de os microrganismos
formarem biofilme aumentou significativamente a resistência aos fármacos, dificultando
o tratamento. Nestes casos, as opções terapêuticas são a remoção do tecido ou implante
infectado ou combinar e até aumentar a dose de fármaco administrada. Em ambos os
casos pode haver consequências negativas relacionadas ao aumento do tempo e dos
custos de hospitalização, bem como a uma sobrecarga renal provocada por altas doses
de fármacos e aumento da morbi-mortalidade. Por isso, a busca por novas estratégias e
tecnologias para o combate destas infecções tem sido alvo importante na pesquisa. O
monolaurato de glicerol é usado na indústria farmacêutica e de alimentos como agente
emulsificante. Apresenta ação antimicrobiana, porém, sua baixa solubilidade em água
dificulta seu uso como alternativa terapêutica em decorrência da baixa biodistribuição.
Neste contexto, a nanotecnologia têm mostrado resultados promissores aumentando a
solubilidade e biodisponibilidade do agente e, assim, alcançando os sítios mais difíceis
da infecção como no caso dos biofilmes. Assim, esse trabalho teve como objetivo
utilizar nanocápsulas contendo monolaurato de glicerol para o tratamento de biofilmes
de bactérias e leveduras, além de verificar aspectos de toxicidade relacionados a esta
terapia. Foram utilizadas nanocápsulas produzidas pelo método de deposição interfacial
do polímero pré-formado. As nanopartículas foram caracterizadas quanto ao diâmetro
médio, índice de polidispersão, potencial zeta, pH e morfologia por microscopia
eletrônica de transmissão que mostraram valores aceitáveis para predizer estabilidade do
sistema. Para os testes microbiológicos foram utilizadas as cepas Pseudomonas
aeruginosa PAO1 e Candida albicans (ATCC 14053). Inicialmente foi feita a
determinação da concentração inibitória e bactericida/fungicida mínima. Foram
realizados ensaios de quantificação do biofilme, curva de crescimento além de
microscopia de fluorescência e de força atômica. Para os testes de toxicidade, foram
usadas linhagens celulares como células VERO, mononucleares de sangue periférico e
eritrócitos. Foram realizados teste de viabilidade, dosagem da enzima Lactato
desidrogenase, teste das substâncias reativas ao ácido tiobarbitúrico (TBARS),
percentual de hemólise e ensaio cometa. Além dos ensaios in vitro foi realizado um
ensaio in vivo de toxicidade com peixes Rhamdia quelen. Os testes iniciais mostraram
que as nanopartículas foram capazes de inibir o crescimento microbiano em uma
concentração menor quando comparado com o monolaurato de glicerol livre. Os ensaios
antibiofilme mostraram redução de aproximadamente 50% do biofilme tratado com as
nanopartículas contendo monolaurato de glicerol. Além disso, o tratamento com as
nanopartículas eliminou quase totalmente o biofilme em 48 horas enquanto o
monolaurato na forma livre não causou efeito. Os ensaios de viabilidade demonstraram
que o monolaurato de glicerol livre possui um importante efeito citotóxico, enquanto as
nanocápsulas mostraram efeito protetor. O monolaurato nanoestruturado demonstrou
redução nos danos celulares em ensaios como liberação de Lactato desidrogenase,
pesquisa de TBARS e porcentagem de hemólise. O ensaio in vivo realizado com peixes
mostrou alta mortalidade causada pela substância na forma livre enquanto as
nanocápsulas demonstraram redução significativa na mortalidade dos animais.
Palavras chave: Biofilmes, nanotecnologia, citotoxicidade, Rhamdia quelen
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ABSTRACT
Biofilms are microbial clusters surrounded by a matrix of extracellular polysaccharides.
In the 21st century it has been proven that the ability of microorganisms to form biofilm
significantly increased drug resistance, making treatment difficult. In these cases, the
therapeutic options are the removal of the infected tissue or implant or to combine and
even increase the dose of drug administered. In both cases, there may be negative
consequences related to increased hospitalization time and costs, as well as renal
overload caused by high doses of drugs and increased morbidity and mortality.
Therefore, the search for new strategies and technologies to combat these infections has
been an important target in the research. Glycerol monolaurate is used in the
pharmaceutical and food industry as an emulsifying agent. It presents antimicrobial
action, however, its low solubility in water makes difficult its use as a therapeutic
alternative due to low biodistribution. In this context, nanotechnology has shown
promising results increasing the solubility and bioavailability of the agent and thus
reaching the most difficult sites of infection as in the case of biofilms. Thus, the
objective of this work was to use nanocapsules containing glycerol monolaurate for the
treatment of biofilms of bacteria and yeasts, as well as to verify aspects of toxicity
related to this therapy. Nanocapsules produced by the interfacial deposition method of
the preformed polymer were used. The nanoparticles were characterized in terms of the
mean diameter, polydispersity index, zeta potential, pH and transmission electron
microscopy morphology, which showed acceptable values to predict system stability.
Pseudomonas aeruginosa strains PAO1 and Candida albicans (ATCC 14053) were
used for the microbiological tests. Initially the minimum inhibitory and bactericidal /
fungicidal concentration determination was made. Biofilm quantification, growth curve,
and fluorescence and atomic force microscopy tests were performed. For toxicity tests,
cell lines were used as VERO cells, peripheral blood mononuclear cells, and
erythrocytes. A viability test, lactate dehydrogenase enzyme test, thiobarbituric acid
reactive substance test (TBARS), the percentage of hemolysis and comet assay was
performed. In addition to the in vitro assays, an in vivo toxicity test with Rhamdia
quelen fish was performed. Initial tests showed that the nanoparticles were able to
inhibit microbial growth in a lower concentration when compared to the free glycerol
monolaurate. The antibiofilm assays showed approximately 50% reduction of the
biofilm treated with the nanoparticles containing glycerol monolaurate. In addition,
treatment with the nanoparticles almost completely eliminated the biofilm in 48 hours
while the monolaurate in the free form had no effect. The viability assays demonstrated
that the free glycerol monolaurate has an important cytotoxic effect, while the
nanocapsules showed a protective effect. Nanostructured monolaurate demonstrated a
reduction in cell damage in assays such as lactate dehydrogenase release, TBARS
detection, and hemolysis percentage. The in vivo assay performed with fish showed
high mortality caused by the substance in the free form while the nanocapsules
demonstrated a significant reduction in the mortality of the animals.
Keywords: Biofilms, nanotechnology, cytotoxicity, Rhamdia quelen
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Sumário
1 INTRODUÇÃO ............................................................................................ 7
1.1 JUSTIFICATIVA ................................................................................... 8
1.2 OBJETIVOS ........................................................................................... 9
1.3 INTERDISCIPLINARIDADE ............................................................... 9
1.4 ORGANIZAÇÃO DA TESE ................................................................ 10
2 REVISÃO BIBLIOGRÁFICA .................................................................. 12
2.1 BIOFILMES ......................................................................................... 12
2.2 MONOLAURATO DE GLICEROL .................................................... 15
2.3 NANOTECNOLOGIA ......................................................................... 17
3 RESULTADOS ........................................................................................... 19
3.1 CAPÍTULO I: NANOCÁPSULAS CONTENDO MLG: EFEITO EM
BIOFILMES. ...................................................................................................... 20
3.1.1 ARTIGO 1: Nanocapsules with glycerol monolaurate: effects on
Candida albicans biofilm ................................................................................... 20
3.1.2 ARTIGO 2: Characterisation and anti-biofilm activity of glycerol
monolaurate nanocapsules against Pseudomonas aeruginosa ........................... 45
3.2 CAPÍTULO II: TOXICIDADE IN VITRO E IN VIVO DAS
NANOCÁPSULAS DE MLG. ........................................................................... 78
3.2.1 ARTIGO 3: Biocompatibility of glycerol monolaurate nanocapsules:
in vitro cytotoxic studies ..................................................................................... 78
3.2.2 ARTIGO 4:Ecotoxicology of Glycerol Monolaurate nanocapsules 106
4 DISCUSSÃO ............................................................................................. 114
5 CONCLUSÕES ........................................................................................ 115
REFERÊNCIAS .............................................................................................. 117
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1 INTRODUÇÃO
Aproximadamente 70% dos agentes infecciosos de origem hospitalar no Brasil
são resistentes a pelo menos um agente antimicrobiano. Além disso, estima-se que 80%
de todas as infecções em humanos, estejam relacionadas com a formação de biofilmes
(RICHARDS; REED; MELANDER, 2008). O termo biofilme descreve a adesão
irreversível de comunidades mono ou polimicrobianas (bactérias, fungos, protozoários,
algas e vírus) em superfícies biológicas ou sintéticas como implantes, tecidos vivos,
válvulas, ossos, dentes e vários dispositivos médicos (DE LA FUENTE-NÚÑEZ et al.,
2013; GAO et al., 2011; MARTIN et al., 2013).
Estes aglomerados microbianos rapidamente produzem uma matriz polimérica
extracelular que dificulta a penetração de agentes microbianos aumentando a resistência
a estes fármacos. Além disso, os biofilmes geralmente se encontram em sítios de difícil
acesso, tornando raramente efetivos os tratamentos atuais. Hoje eles são responsáveis
pela maioria das infecções microbianas e a melhor opção de combate, além da
prevenção, é a remoção do implante ou tecido colonizado, gerando custos hospitalares e
diminuindo a qualidade de vida do paciente (CHEN et al., 2014; FORIER et al., 2014;
TAMILVANAN; VENKATESHAN; LUDWIG, 2008).
O monolaurato de glicerol (MLG) é uma substância amplamente utilizada como
emulsificante na indústria alimentícia e cosmética. Possui atividade antimicrobiana
contra diversos cocos Gram positivos, incluindo Bacillus anthracis, Staphylococcus
aureus, Streptococcus sp. (SCHLIEVERT et al., 1992; VETTER; SCHLIEVERT,
2005) e algumas bactérias Gram negativas (CARPO; VERALLO-ROWELL;
KABARA, 2008). Em forma de gel, o MLG inibiu o crescimento vaginal de Candida
albicans e microrganismos causadores de vaginose bacteriana (Gardnerella vaginalis),
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sem inibir as bactérias benéficas para a microbiota vaginal (STRANDBERG et al.,
2010). Seu uso como antimicrobiano sistêmico é pouco explorado devido a
características físico-químicas do MLG, como a baixa solubilidade em água e o alto
ponto de fusão (LOPES et al., 2016a).
A nanotecnologia pode melhorar aspectos de solubilidade de substâncias,
potencializar a atividade antimicrobiana e diminuir os efeitos adversos devido à redução
da dose (BHAWANA et al., 2011). A nanotecnologia é uma das ferramentas mais
promissoras para contribuir com o tratamento das infecções microbianas (CAVALIERI
et al., 2014; ZHU et al., 2014). Dentre as áreas, destaca-se a produção de nanopartículas
(NP’s) para o potencial anti-biofilme que mostrou resultados importantes para a
indústria farmacêutica (KROLL et al., 2009; MARKOWSKA; GRUDNIAK;
WOLSKA, 2013; MU et al., 2016; QAYYUM; KHAN, 2016; SAHARAN et al., 2013).
1.1 JUSTIFICATIVA
Os biofilmes contribuem para as infecções associadas ao uso de cateter, que nos
Estados Unidos causam aproximadamente 10.000 mortes e mais de 11 bilhões de
dólares em custos hospitalares por ano. Estima-se que dos 5 milhões de cateteres
urinários inseridos em pacientes por ano, em aproximadamente 20% ocorre a formação
de biofilmes (ANDERSON et al., 2003; SCHACHTER, 2003) e pacientes em
hemodiálise são comumente afetados pela formação do biofilme. Além disto, as
infecções crônicas como endocardite, otite média, pneumonia, fibrose cística e
infecções associadas à biomateriais implantados, frequentemente estão relacionados
com infecções associadas a biofilme (COSTERTON; STEWART; GREENBERG,
1999). Portanto, a busca por novas opções terapêuticas tem sido foco frequente nas
pesquisas: não somente o desenvolvimento de novos fármacos, mas também novos
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materiais contendo nanoestruturas estão apresentando resultados promissores na
atividade antibiofilme. Neste contexto, o MLG apresenta grande potencial terapêutico
para problemas decorrentes a biofilme e é de grande interesse a avaliação da atividade
antimicrobiana desta substância na forma nanoestruturada.
1.2 OBJETIVOS
1.2.1 Objetivo geral
Avaliar o potencial antibiofilme e os efeitos tóxicos de Nanocápsulas contendo
MLG (NMLG).
1.2.2 Objetivos específicos
- Avaliar in vitro a atividade antibiofilme das NMLG e comparar com o MLG na
forma livre;
- Avaliar in vitro a atividade citotóxica das NMLG contra diferentes linhagens
celulares como: células VERO, células mononucleares de sangue periférico e
eritrócitos;
- Determinar in vivo a atividade tóxica do MLG e NMLG frente a peixes
(Rhamdia quelen);
1.3 INTERDISCIPLINARIDADE
A nanotecnologia tem como objetivo a produção, caracterização e aplicação de
novas estruturas, materiais e sistemas, com forma e tamanho, em uma escala
nanométrica. Esta área, diferente das demais que utilizam disciplinas específicas, é
considerada interdisciplinar devido a sua abrangência (KIM; RUTKA; CHAN, 2010).
Para chegar a este objetivo, é necessária a união de áreas como biologia, química, física,
matemática entre outras. Conhecimentos da física são necessários para entender o
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comportamento e propriedades das nanopartículas em determinados ambientes e
situações, além da compreensão do princípio envolvido nos equipamentos para
caracterização dos nanomateriais. A química se faz necessária para esclarecer as
diversas interações químicas das formulações e os aspectos de solubilidade dos
produtos.
A utilização de ferramentas estatísticas requer conhecimento da área matemática e
informática para melhor demonstrar os resultados obtidos. Para este estudo,
conhecimentos de biologia e veterinária, se mostram importantes principalmente nos
ensaios realizados com peixes, uma vez que deve ser mantido um ambiente adequado
para que os ensaios in vivo sejam confiáveis. Para os ensaios microbiológicos e
citotóxicos são necessários conhecimentos na área biológica/biomédica. Deste modo, o
caráter interdisciplinar do Programa de Pós-Graduação em Nanociências, tornou e torna
possível a realização do presente trabalho.
1.4 ORGANIZAÇÃO DA TESE
Nesta tese as metodologias e os resultados produzidos estão organizados em dois
capítulos. O primeiro capítulo trata da atividade antimicrobiana (composto por dois
artigos) e o segundo capítulo trata dos estudos de toxicidade (composto por dois
artigos).
O primeiro capítulo apresentado é NANOCÁPSULAS CONTENDO MLG:
EFEITO EM BIOFILMES. Este capítulo contem o artigo 1: NANOCAPSULES WITH
GLYCEROL MONOLAURATE: EFFECTS ON CANDIDA ALBICANS BIOFILMS
publicado no periódico científico Microbial Pathogenesis (Qualis CAPES
Interdisciplinar B1, fator de impacto 2,332). Onde foi avaliado o potencial antibiofilme
das nanopartículas em biofilmes formados in vitro pelo fungo Candida albicans.
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No mesmo capítulo também está apresentado o artigo 2:
CHARACTERISATION AND ANTI-BIOFILM ACTIVITY OF GLYCEROL
MONOLAURATE NANOCAPSULES AGAINST PSEUDOMONAS AERUGINOSA
publicado no periódico científico Microbial Pathogenesis (Qualis CAPES
Interdisciplinar B1, fator de impacto 2,332). No artigo 2 estão apresentados resultados
da nanocápsula contendo MLG sobre biofilmes e a influência sobre fatores de virulência
da bactéria P. aeruginosa.
No segundo capítulo intitulado TOXICIDADE IN VITRO E IN VIVO DAS
NANOCÁPSULAS CONTENDO MLG estão resultados de dois artigos. O artigo 3
intitulado GLYCEROL MONOLAURATE NANOCAPSULES FOR BIOMEDICAL
APPLICATIONS: IN VITRO TOXICOLOGICAL STUDIES foi aceito no periódico
Naunyn-Schmiedeberg's Archives of Pharmacology (Qualis CAPES Interdisciplinar A2,
fator de impacto 2,238). O artigo mostra resultados dos efeitos das nanocápsulas e do
MLG livre frente às culturas celulares.
No capítulo 2 também está apresentado o resultado referente ao ensaio de
toxicidade das nanopartículas e da substância na forma livre frente a peixes da espécie
Rhamdia quelen que juntamente com outros resultados paralelos foi publicado no
periódico Ecotoxicology and Environmental Safety (Qualis CAPES Interdisciplinar A1,
fator de impacto: 3,974).
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2 REVISÃO BIBLIOGRÁFICA
2.1 BIOFILMES
Os microrganismos são estruturas presentes em diversos habitats, porém são
capazes de desenvolver complexos e variados comportamentos (COSTERTON;
STEWART; GREENBERG, 1999). Na forma livre (planctônica), os microrganismos
encontram-se em suspensão e vivem isoladamente, enquanto que, na forma séssil, se
encontram aderidos a superfícies sob a forma de biofilmes (STOODLEY et al., 2002).
O biofilme pode ser definido como uma comunidade complexa de microrganismos
aderida a uma superfície biótica ou abiótica envolvida por uma matriz polimérica,
produzida por eles mesmos como uma forma de proteção às defesas do hospedeiro e aos
agentes terapêuticos (DUNNE, 2002). Há várias vantagens para os microrganismos
quando estão na forma de biofilme se comparados aos seus homólogos de vida livre.
Essas vantagens ocorrem devido ao fato dos agregados de microrganismos apresentarem
maior disponibilidade de nutrientes, interferindo nas taxas de crescimento, cooperação
metabólica e proteção aos fatores externos (BEHLAU; GILMORE, 2008).
Os biofilmes podem ser constituídos por uma única espécie, ou por comunidades
derivadas, formadas por várias espécies bacterianas, fungos, leveduras, algas e outros
organismos celulares (polimicrobiano) (SAUER; RICKARD; DAVIES, 2007). Quando
ocorre o crescimento de biofilme polimicrobiano, uma espécie pode ser favorecida pela
presença da outra em uma interação chamada de comensalismo, melhorando a
degradação de compostos orgânicos em comparação com as monoculturas
(NIKOLAEV; PLAKUNOV, 2007). No entanto, os microrganismos representam menos
de 10% do biofilme (SATPATHY et al., 2016). Os biofilmes são compostos também
pelas substâncias poliméricas extracelulares (EPS) ou matriz exopolissacarídica e por
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quaisquer outros resíduos do ambiente colonizado, além de proteínas, lipídeos, DNA,
RNA, íons e água, formando uma estrutura porosa e altamente hidratada (BEHLAU;
GILMORE, 2008).
Os exopolissacarídeos constituídos por diferentes biopolímeros são os principais
componentes que determinam a estrutura e a integridade funcional do biofilme (KIVES;
ORGAZ; SANJOSÉ, 2006). São responsáveis por até 90% da massa do biofilme e
oferecem um ambiente protetor às células microbianas, dificultando a penetração de
agentes antimicrobianos. Assim, agem como uma barreira de filtragem, e ocasionam
uma penetração lenta ou reduzida de agentes antimicrobianos em geral. A matriz
também protege os microrganismos contra a dessecação, oxidação, radiação ultravioleta
e defesa imunológica do hospedeiro (FLEMMING; WINGENDER, 2010).
O biofilme inicia com a aderência microbiana à superfície, e este processo é
condicionado à interferência de fatores biológicos (como o crescimento das células
microbianas e sua divisão, produção e excreção de EPS) e fatores não biológicos. Entre
os fatores não biológicos são reconhecidas as interações químicas como as forças de
Van der Walls, interações hidrofóbicas, eletrostáticas e ligações de hidrogênio que
ocorrem entre as macromoléculas. Este estágio inicial de adesão, em que há
envolvimento de interações físico-químicas entre as superfícies, designa-se adesão
primária (WATNICK; KOLTER, 2000). Em um segundo estágio, os microrganismos
são levemente aderidos a superfície, induzindo distintas fases de crescimento e intensa
divisão celular (FLEMMING et al., 2000). O processo de adesão é concretizado com a
produção de exopolissacarídeos, formando complexos com os materiais da superfície na
qual aderem e/ou através de receptores específicos localizados na superfície das paredes
celulares (caracterizando a adesão secundária) (SUTHERLAND, 1997). Em seguida
inicia o processo de maturação do biofilme, formado por microrganismos que são
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interceptados por canais de água que permitem a entrada dos nutrientes. Ao final deste
processo, o biofilme atinge uma massa crítica, sendo estabelecido um equilíbrio
dinâmico no qual o crescimento de células é compensado pela liberação de células
planctônicas disponíveis para a colonização de outras superfícies formando novos
biofilmes (Figura 1) (COSTERTON; MONTANARO; ARCIOLA, 2005; DUNNE,
2002).
Figura 1: Processo de formação do biofilme. Adesão do microrganismo à superfície (1),
produção de exopolissacarídeo (2), maturação (3,4) e alcance de massa crítica liberando células
bacterianas para se aderirem em outros locais e formarem novos biofilmes (5).
(Adaptado de MONROE, 2007).
Uma vez instalado o biofilme como uma estratégia de sobrevivência para os
microrganismos, este então fornece vantagens importantes como menor exposição a
carências nutricionais, radicais de oxigênio e antimicrobianos; mudanças de pH; abrigos
de predação; manutenção das atividades de enzimas extracelulares, além de ocorrer uma
resistência aumentada à fagocitose (FUCHS et al., 2010; FUX et al., 2005; KEREN et
al., 2004; O’GARA, 2007).
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2.2 MONOLAURATO DE GLICEROL
O Monolaurato de Glicerol (MLG) é uma substância que apresenta atividade
frente a bactérias, fungos e vírus, encontrado em baixas concentrações no óleo de coco e
no leite materno (ANANG et al., 2007; HORNUNG; AMTMANN; SAUER, 1994). É
reconhecido pela Food and Drug Administration (FDA) como sendo seguro para uso
oral e frequentemente utilizado na indústria alimentícia como emulsificante
(considerado não tóxico mesmo em concentrações elevadas). Atua como um
conservante, impedindo a contaminação e deterioração de alimentos e cosméticos por
microrganismos (BAUTISTA et al., 1993; RAZAVI-ROHAN; GRIFFITHS, 1994).
A estrutura do MLG é composta por um monoéster de glicerol e um ácido graxo
chamado ácido láurico como pode ser visto na Figura 2. O monolaurato é a estrutura
mais estudada entre os monoésteres de ácido graxo com propriedades antimicrobianas.
Entretanto as propriedades físico-químicas do MLG incluem alto ponto de fusão e baixa
solubilidade em água, glicerol e outros solventes tornando difícil a sua utilização como
agente antimicrobiano.
Figura 2: Estrutura Química do Monolaurato de Glicerol
Fonte: Elaborado pelo autor.
Já se conhece o potencial antibacteriano do MLG frente a bactérias Gram
positivas (PROJAN et al., 1994; SCHLIEVERT et al., 1992), porém apresentam pouca
atividade em Lactobacillus sp. (protetores da mucosa vaginal). O uso de MLG como gel
intravaginal para o combate de C. albicans e Gardnerella vaginalis (agentes etiológicos
da candidíase e vaginose bacteriana) também já foi descrito (STRANDBERG et al.,
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2010). Algumas bactérias Gram negativas, Enterobactérias não são suscetíveis ao MLG
(SCHLIEVERT et al., 1992). Além disso, nosso grupo de estudos mostrou a atividade
do monolaurato de glicerol frente ao Paenibacillus larvae agente causador da American
Foulbrood Disease que acomete abelhas (LOPES et al., 2016a).
Em um estudo realizado por Carpo e colaboradores (2008) os autores buscaram
verificar a suscetibilidade de diferentes microrganismos frente ao MLG e comparar seus
efeitos com antimicrobianos comumente usados na prática clínica. Os resultados
mostraram uma significativa sensibilidade in vitro de patógenos isolados de infecção de
pele como Staphylococcus sp., Streptococcus sp. e Klebsiella sp. Todos os
microrganismos apresentaram resistência a fármacos como penicilina, eritromicina e
ácido fusídico, porém, nenhum apresentou resistência ao MLG.
O MLG diferentemente da maioria dos antimicrobianos, que atingem um único
alvo, atua em vários sistemas de sinalização bacteriana. Atua inicialmente interagindo
com a membrana plasmática, (SCHLIEVERT et al., 1992; VETTER; SCHLIEVERT,
2005) afetando a transdução do sinal e a captação de aminoácidos (KABARA;
MARSHALL, 2005). Também atua impedindo a produção de enzimas e fatores de
virulência como proteína A, α-hemolisina e β-lactamases (RUZIN; NOVICK, 1998).
Em partículas virais o MLG atua contra vírus envelopados, incluindo vírus Influenza e
Herpes vírus, interferindo na fusão do vírus com as células, além de prevenir o processo
inflamatório (necessário para a penetração na superfície da mucosa) (THORMAR et al.,
1987). Além disto, o MLG demonstrou capacidade de inibição da produção da síndrome
do choque tóxico causada por toxinas produzidas por Staphylococcus aureus. Essa
síndrome é caracterizada por febre alta, pressão baixa, erupção cutânea e descamação da
pele (CHESNEY, 1989; DAVIS et al., 1980).
17
2.3 NANOTECNOLOGIA
Em 1959, no Instituto de Tecnologia da Califórnia, o pesquisador Richard
Feynman realizou uma palestra no encontro American Physical Society falando sobre a
construção de nanoestruturas, molécula por molécula, átomo por átomo. Surgia a
nanotecnologia e a manipulação de materiais em escala atômica modificando as
propriedades físicas e químicas e facilitando a penetração em barreiras e membranas
celulares (MANSOORI; FAUZI SOELAIMAN, 2005; YANG; PETERS; WILLIAMS,
2008).
A nanotecnologia abrange diversas áreas como Física, Química, Medicina,
Biologia, Informática entre outras, e isso a torna um campo interdisciplinar. Usando a
nanotecnologia, é possível determinar propriedades e o modo de liberação de fármacos
conjugados a nanoestruturas. Dentre as nanoestruturas mais utilizadas para este fim
estão as nanopartículas (poliméricas ou lipídicas), os nanotubos, pontos quânticos,
dendrímeros e os lipossomas (PUTHETI; OKIGBO; SAI, 2008; VAUTHIER et al.,
2003).
Os compostos bioativos nanoencapsulados representam uma alternativa para
aumentar a estabilidade da substância ativa, isso porque protege o fármaco de interações
indesejáveis. Para agentes antimicrobianos, a nanoencapsulação pode aumentar a
concentração do fármaco na região onde os microrganismos se localizam (WEISS et al.,
2009). Substâncias como o MLG, que possui atividade antimicrobiana, mas devido aos
problemas de solubilidade, permanece inviável o uso terapêutico (KABARA;
MARSHALL, 2005). Por esse motivo, a nanoestruturação desta substância, pode vir a
ser uma alternativa viável para os problemas usuais e vir a se tornar um aliado no
combate a infecções.
18
Para reduzir a carga do tratamento, desenvolver uma formulação com uma
liberação controlada do fármaco, seria benéfico já que a liberação gradual faz com que o
fármaco permaneça mais tempo no organismo mesmo utilizando uma concentração
baixa. Estudos com lipossomas de ciprofloxacina mostram que a formulação age
somente no local de ação tornando-se um ótimo tratamento para infecções respiratórias.
Essa melhor biodistribuição proporciona uma maior concentração do fármaco
exclusivamente no local de ação, reduzindo assim efeitos tóxicos e adversos além de
contornar o problema da resistência bacteriana (BAKKER-WOUDENBERG et al.,
2001; GUBERNATOR et al., 2007).
19
3 RESULTADOS
Esta investigação alcançou resultados significativos frente às ações e objetivos
assumidos na Introdução e explicados no Item 3 - Metodologia. Esses resultados podem
ser visualizados em artigos publicados.
20
2.1 CAPÍTULO I: NANOCÁPSULAS CONTENDO MLG: EFEITO EM
BIOFILMES.
3.1.1 ARTIGO 1: NANOCAPSULES WITH GLYCEROL MONOLAURATE:
EFFECTS ON CANDIDA ALBICANS BIOFILM
O primeiro resultado apresentado foi referente à atividade das nanocápsulas
contendo MLG frente a biofilmes de Candida albicans. Neste trabalho foram tratados:
Produção e caracterização das nanocápsulas contendo MLG;
Determinação da concentração inibitória mínima da formulação e da substância
na forma livre frente ao fungo Candida albicans pela técnica de microdiluição;
Curva de crescimento;
Avaliação da atividade contra o biofilme formado pelas técnicas de cristal
violeta e Calcofluor White;
Curva de atividade anti-biofilme;
Avaliação do potencial para prevenir a formação de biofilme;
21
Nanocapsules with glycerol monolaurate: effects on Candida albicans biofilms
Leonardo Quintana Soares Lopes 1,2 *
, Cayane Genro Santos 2, Rodrigo de Almeida
Vaucher1,2
, Renata Platcheck Raffin2, Roberto Christ Vianna Santos
1,2,3
1 Laboratory of Microbiology Research, Centro Universitário Franciscano, Santa Maria,
Brazil
2 Post Graduate Program in Nanosciences, Centro Universitário Franciscano, Santa
Maria, Brazil
3 Microbiology and Parasitology Department, Health Sciences Center, Universidade
Federal de Santa Maria, Santa Maria, Brazil
*Correspondent author e-mail address: [email protected]
Permanent address: Centro Universitário Franciscano, Laboratory of Microbiology
Reserach
Rua dos Andradas 1614, Santa Maria-RS, Zip Code 97010-032, Brazil
Abstract
Candida albicans does not only occur in the free living planktonic form but also grows
in surface-attached biofilm communities. Moreover, these biofilms appear to be the
most common lifestyle and are involved in the majority of human Candida infections.
Nanoparticles can be used as an alternative to conventional antimicrobial agents and can
also act as carriers for antibiotics and other drugs. In view of this, the aim of the study
was develop, characterize and verify the anti-biofilm potential of GML Nanocapsules
against C. albicans. The GML Nanocapsules showed mean diameter of 193.2 nm,
mailto:[email protected]
22
polydispersion index of 0.044, zeta potential of -23.3 mV and pH 6.32. The
microdilution assay showed MIC of 15.5 µg mL-1
to GML Nanocapsules and 31.25 µg
mL-1
to GML. The anti-biofilm assay showed the significantly reduction of biomass of
C. albicans biofilm treated with GML Nanocapsules while the GML does not exhibit
effect. The kinetic assay demonstrated that at 48 hours, the GML Nanocapsules reduce
94% of formed biofilm. The positive results suggest the promisor alternative for this
public health problem that is biofilm infections.
Keywords: Glycerol monolaurate; Nanocapsules; anti-biofilm; kinetic; Candida
albicans.
1. Introduction
Biofilms are compact bacterial clusters that can adhere to many surfaces. They rapidly
produce an extracellular polymeric matrix that is hard to penetrate, thus increasing the
resistance of therapeutic drugs. Moreover, they are usually localized at sites difficult to
reach, so current treatments are rarely successful. Currently, they are responsible for
most microbial infections and the best option, besides prevention, is to remove the
colonized tissue or implant [1–3].
The biofilm infections in hospital environment are a serious problem of public health
and many methods has been used to try minimize or eliminate them. The great difficult
lies in the fact of that many of these methods have important disadvantages, because
lead to clinical complications and develop strains multi resistant [4]
Nanotechnology is one of the most prominent areas with the potential to tackle almost
every aspect of microbial infections [2,5,6]. One of the main areas in focus is the
development of therapeutic nanoparticles (NPs) for anti-biofilm applications. NPs can
be synthesized through many different methods and approaches [7]. The reason why
23
these molecules are so well studied and tested in the therapeutics of infections lies in
their properties. Recently, this theme has been reviewed focusing on liposome and
polymeric nanoparticles [1]
The glycerol monolaurate (GML) is a natural compound recognized as safe by The
Food and Drug Administration (FDA). The antimicrobial potential of GML against
many Gram Positive coccus in addition to Bacillus anthracis is known [8]. A previous
study performed by Schlievert and Peterson, showed the ability of GML to inhibit the
biofilm formation of three strains of Staphylococcus aureus including Methicillin
resistant Staphylococcus aureus (MRSA) [9]. The present work is the first study that
associates GML nanoparticles and biofilm, that despite promising, the use of GML is
not expanded due the low solubility in water, leading to low bioavailability. The aim of
present study was for the first time develop and characterize GML nanoparticles aiming
the application on Candida albicans biofilms.
2. Materials and methods
2.1 Materials
The GML was purchased by Seebio Biotech, Inc®
, Xangai, China. Sorbitan monooleate
(Spam 80®
), polysorbate 80 (Tween 80®
) and acetone was purchased from Labsynth®
(São Paulo, Brazil); capryc/caprylic triglyceride mixture was acquired from Brasquim
(Porto Alegre, Brazil); the polymeric blende PMMA/PEG was supplied by Laboratory
of Nanotechnology of Centro Universitário Franciscano (Santa Maria, Brazil).
2.2 Glycerol monolaurate nanocapsules
The GML Nanocapsules were produced according to the method described previously
[10] with modifications. The aqueous phase was prepared with polysorbate 80 (0.194 g)
24
and purified water (134 mL) at 40°C under moderated stirring. In the organic phase, the
GML (0.25 g) was solubilized with sorbitan monooleate (0.194 g), capryc/caprylic
triglyceride (0.8 g), and polymeric blende PMMA-PEG (0.25 g) in acetone (67 mL) at
40°C under moderated magnetic stirring. After solubilized, the organic phase was
poured into de aqueous phase under magnetic stirring, being maintained for 10 minutes.
The organic solvent and the water were evaporated in rotatory evaporator (Fisatom®
Brazil) to adjust concentration to 1 mg/mL getting 25 mL of formulation. A blank
formulation (Blank Nanocapsules) was developed in the same way as GML
Nanocapsules (but without GML).
2.3 Characterization of GML Nanocapsules
After preparation, the formulations were characterized as size and polydispersity index
(PDI) by dynamic light scattering (DLS), zeta potential by electrophoresis in a Zetasizer
Nano-ZS (Malvern Instruments, United Kingdom) and the pH was evaluated using
potentiometer (Digimed®
). Each parameter was evaluated in triplicated (n=3) and
results were expressed by average ± standard deviation (SD). The morphology of the
nanocapsules were analyzed by transmission electron microscopy operating at 80 kV
(TEM; Jeol, JEM 1200 Exll, Japan). Diluted suspensions (1:10 v/v in water) were
deposited on specimen grid (Formvar-Carbon support films), negatively stained with
uranyl acetate solution (2% w/v) and observed at different magnifications.
2.4 Microorganism
The strain Candida albicans (ATCC 14053) was obtained by American Type Culture
Collection. This microorganism was maintained on culture medium with glycerol and
cooled at -80 °C. The sample was unfrozen, inoculated on Brain Heart Infusion broth
25
(BHI) and incubated for 24 hours. After, it were seeded on Sabouraud agar and
incubated for 24 hours at 37 °C.
2.5 Minimal Inhibitory Concentration (MIC) and Minimal bactericidal
concentration (MBC)
The MIC was performed by microdilution method on 96-well plate [11]. Different
concentrations GML and GML Nanocapsules were add on wells containing Mueller
Hinton broth (MHB). The positive control was considered the well with inoculum in
MHB and negative control only MHB with saline. The assay was performed in
triplicate. After the process, the plate was incubated to 24 hours at 37 °C. After
incubation, the assay was revealed with 2,3,5 triphenyl tetrazolium chloride. To
determine the MBC, an aliquot of 1µl was taken of each well, seeded on Sabouraud agar
plate and incubated to 24 hours. After, the colonies were identified and the lowest
concentration which does not demonstrated microbial growth was considered the MBC.
2.6 Effect of GML and GML Nanocapsules on microbial growth
Microbial growth curve was observed by inoculating the 96 well-plate with Mueller
Hinton broth containing 1.5 x 108 CFU/mL of C. albicans and loaded with different
concentrations of GML and GML nanocapsules (3.9 – 62.5 µg/mL). The plate was
incubated at 37 °C for 30 hours and the absorbance was reader at 600 nm [12].
2.7 Biofilm formation
The biofilm was formed according to the conditions previously optimized and described
[13,14] with modifications. Fresh, exponentially grown culture of C. albicans was
diluted to be 106 CFU/mL and 50 µL was added to flat-bottomed 24-well plates
26
(Nunclon™ D surface, Nunc, Roskilde, Denmark), containing 500 µL of BHI broth and
the plate was incubated in 37 °C, for 24 hours.
2.8 Efficiency of GML and GML Nanocapsules against biofilm developed
After formation of biofilm, it was performed the treatment and incubated for 24 hours in
condition of 37 °C according to Manner et al. [15]. The treatment was performed with
500 µL of a suspension containing 1 mg/mL of GML or GML Nanocapsules. A positive
control was performed containing only BHI broth and the C. albicans strain while the
negative control was just BHI broth.
2.9 Quantification of biofilm biomass
After the treatment, the supernatant was removed and washed four times with PBS and
them, it was performed the quantifications. The result of biofilm treatment was
measured fixing with 95% of methanol and staining with 500 µL of 0.1% of crystal
violet or 1% of safranin for 10 min at room temperature (RT). After incubation, the
well-plates were washed with PBS and photos (Fig 4) were taken. Ethanol 95% was
added to dissolve the coloring and after, transferred into other plate to measure
spectrophotometrically at 570 nm to crystal violet and 492 to safranin in
spectrophotometer (TP-Reader; ThermoPlate, Goiás, Brazil). The biofilm formation was
determined by the difference between the mean OD readings obtained in the positive
control (BHI broth and C. albicans strain) and the treatment with GML and GML
nanocapsules.
2.10 Efficiency against biofilm formation
The GML and GML nanocapsules were tested to verify the ability in prevent the
biofilm formation of C. albicans. The assay was performed in three replicates on 96
27
well-plates. It were used three sub inhibitory concentrations (0.5 x MIC, 0.25 x MIC
and 0.125 x MIC) which were added together with microorganism. The experiment was
performed in BHI broth for 24 hours. After incubation, the biofilm was revealed such
item above. Only BHI broth with C. albicans was considered control and the percentage
of inhibition was calculated by OD Test / OD Control x 100.
2.11 Kinetics of anti-biofilm activity on developed biofilm
Efficacy of GML and GML Nanocapsules were evaluated against C. albicans biofilm
by the time-dependent killing assay. Biofilms of C. albicans were formed in microtube
and treated with 1× MBC of compound or formulation. Over a series of time intervals
of 0, 3, 6, 12, 24 and 48 hours, the anti-biofilm activity was measured with the safranin
stain assay and the absorbance was reader in spectrophotometer (TP-Reader;
ThermoPlate, Goiás, Brazil) at 492 nm. After coloration, the microtubes were washed 3
times with PBS, and was used ethanol to dissolve the safranin adhered in microtube.
After discoloration, 300 µL were transferred to microplate for reading. The assay was
performed in 3 replicates.
2.12 Biofilm treatment on glass slide and stained with Calcofluor White
A glass slide was inserted into petri dish, containing 10 mL of BHI broth. After, 50 µL
of suspension containing C. albicans was added into the plate. The plate was incubated
at 37 °C for 48 hours. After biofilm formation, was performed the treatment with 1x
MBC of GML (62.5 µg/mL) and GML Nanocapsules (31.25 µg/mL). A dish without
treatment was considered the Positive Control (only BHI + C. albicans suspension).
After incubation, the glass slide was removing from dish, washed with PBS and dried at
RT. Three drops of KOH (10%) and 3 drops of Calcofluor White Stain were dispersed
28
in glass slide. After 1 minute at RT, the glass slide was analyzed in Fluorescence
Microscopy to observe the biofilm.
2.13 Statistical analysis
The results of microbiological assays were submitted to One-way analyses of variance
(ANOVA) following by Tukey test with 95% of significance. The experiments were
performed in 3 replicates (n=3) except the biofilm assay which was carried in 2
replicates.
3. Results
3.1 Characterization of GML Nanocapsules
After preparation, the formulation was evaluated as physical-chemical characteristic.
The measurements showed values of pH, mean diameter, polydispersion index and zeta
potential. The values were 6.32 ± 0.31 to pH, mean diameter about 193.2 ± 4 nm,
polydispersion index of 0.044 ± 0.028 and zeta potential of -23.3 ± 3 mV.
The image of nanoparticle obtained by transmission electron microscopy was shown in
Figure 1.
3.2 MIC and MBC
The MIC and MBC can be visualized in Table 1. The Blank Nanocapsules was tested in
the same way with GML Nanocapsules but without antimicrobial activity (data not
shown)
29
3.3 Growth curve
The growth curve of C. albicans showed a difference of inhibition in relation to dose
after 30h of exposition. The result is according to MIC and MBC assays and is
described in Figure 3.
3.4 Biofilm quantification by crystal violet and safranin
After stain procedure, the biofilm was quantified measuring the absorbance and
comparing with positive control (C. albicans + broth). The result was described in
Figure 2. The Blank formulation was tested and do not demonstrate antibiofilm activity
(data not shown)
3.5 Efficiency against formation of biofilm
After absorbance reading, it’s possible observes that GML Nanocapsules inhibit the
formation of biofilm at 0.5 x MIC concentration while the GML does not inhibit the
biofilm formation. The results are described in Figure 5. There was no inhibition of
biofilms treated with Blank Nanocapsules (data not shown)
3.6 Kinetics of inhibition of formed biofilm
After 48 hours, the GML Nanocapsules demonstrated capacity to eliminate virtually all
biofilm (94%), while GML showed a lower effect. The result is described in Figure 5.
3.7 Biofilm treatment on glass slide and stained with Calcofluor White
After staining, the glass slide was observed in Fluorescence microscopy and then took
snapshots. The biofilm was observed in 400 x. The images are demonstrated in Figure
6.
30
4. Discussion
The synthesis of polymeric nanoparticles has been proposed to combat biofilm
infections [16]. The polymeric nanoparticles would function as drug carriers that deliver
the therapeutic molecule into the infected tissue, especially those that are water-
insoluble, improving the effect on the biofilm [16]. The studies of nanoparticles against
biofilm have demonstrated a promissory therapeutic alternative. Some kinds of drug
nanoparticles are able to penetrate the barrier and eliminate biofilm. For example, only
one dose of ciprofloxacin-PLGA nanoparticles reduced the Pseudomonas aeruginosa
biofilm mass, size and live cell density by more than 80%, and repeated administrations
prevented new formations [17]. A study with Melaleuca alternifolia oil nanoparticles
showed anti-biofilm activity of Pseudomonas aeruginosa, also decreased the adhesion
on epithelial cells and impaired the motility of microorganism, while the free oil do not
showed effect [18]. Moreover, the nanoencapsulation of antibiotics resulted in better in
vitro anti-biofilm activity compared to the free antibiotic [19,20].
In the present study, the formulations showed a milky bluish opalescent aspect (Tyndall
effect) demonstrating success on the development [21]. After analysis in transmission
electron microscope, images were produced and it was possible to verify the spherical
shape and nanometric size proving the success of the development of the nanocapsule
(Figure 1).
Negative values to zeta potential are expected when used polymers containing grouping
ester in structure, such as PMMA [22]. High values, in modulus of zeta potential,
indicate that the nanoparticles have charges which allows the repulsion between other
particles preventing the aggregation also predicting the formulation stability [23,24].
The obtained results of characterization in the present study corroborates with works
31
related in literature which use polymeric blende in development of nanoparticles such
carries and suggest homogeneity on size distribution [25–27].
A recent study of our group with GML Nanocapsules showed the antimicrobial activity
against bee pathogen [28].This is the first report which shows GML nanoparticles
against biofilms. The assay with crystal violet demonstrated the high potential of GML
Nanocapsules, against biofilm of C. albicans (Figure 4C), while the GML don’t showed
significant effect being equal to the positive control (Figure 4B and 4A). Studies
performed by Schlievert et al. [9] demonstrated the capacity of GML on inhibit
Staphylococcus aureus biofilms. The GML Nanocapsules demonstrated the ability to
prevent biofilm formation (Figure 5).
In the kinetics assay, the GML Nanocapsules reduced approximately 94% of formed
biofilm on 48 hours while the GML reduced 46% in the same time. In the Calcofluor
stain assay, the GML on concentration of 62.5 µg/mL it was not effective against C.
albicans biofilm. The GML Nanocapsules on concentration of 32.25 µg/mL was able to
significantly reduce the biofilm (Figure 7).
Previous studies with development of nanoparticles with mean size of 220 nm and
260nm were internalized by fungal cells due their reduced size have showed that due
their reduced size, the nanocapsules could be internalized by fungal cells [29,30].
Moreover, the slow release of the GML could have had an important role in anti-biofilm
activity throughout the time of the assay. In addition, a long time of release could help
the dispersion of GML increasing the cellular penetration [31].
5. Conclusion
In conclusion, the study demonstrated the success of development of GML
nanocapsules with acceptable values to predict a stable system. Moreover, the potential
of nanocapsule against C. albicans was higher comparing the free GML. Furthermore,
32
the anti-biofilm activity of GML Nanocapsules showed a therapeutic alternative to
combat biofilms, considering that usual drugs do not penetrate into biofilm matrix and
thus not being effective. Therefore, more studies must be performed to clarify the real
mechanism of action of GML Nanocapsules and the role of nanostructuration on cell
wall.
Conflict of interest statement
We declare that we have no conflict of interest.
Acknowledgment
This work received financial support of PPGPE/Centro Universitário Franciscano-
Probic, CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico),
CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and
FAPERGS (Fundação de Amparo a Pesquisa do Rio Grande do Sul).
References
[1] K. Forier, K. Raemdonck, S.C. De Smedt, J. Demeester, T. Coenye, K.
Braeckmans, Lipid and polymer nanoparticles for drug delivery to bacterial
biofilms, J. Control. Release. 190 (2014) 607–623.
doi:10.1016/j.jconrel.2014.03.055.
[2] F. Cavalieri, M. Tortora, A. Stringaro, M. Colone, L. Baldassarri, Nanomedicines
for antimicrobial interventions, J. Hosp. Infect. 88 (2014) 183–190.
doi:10.1016/j.jhin.2014.09.009.
[3] C.W. Chen, C.Y. Hsu, S.M. Lai, W.J. Syu, T.Y. Wang, P.S. Lai, Metal
nanobullets for multidrug resistant bacteria and biofilms, Adv. Drug Deliv. Rev.
78 (2014) 88–104. doi:10.1016/j.addr.2014.08.004.
[4] H.H. Zadegan, B. Delfan, Evaluation of antibiofilm activity of dentol, Acta Med.
Iran. 47 (2009) 35–40.
[5] C.S. Alves, M.N. Melo, H.G. Franquelim, R. Ferre, M. Planas, L. Feliu, et al.,
Escherichia coli cell surface perturbation and disruption induced by antimicrobial
peptides BP100 and pepR, J. Biol. Chem. 285 (2010) 27536–27544.
doi:10.1074/jbc.M110.130955.
[6] X. Zhu, A.F. Radovic-Moreno, J. Wu, R. Langer, J. Shi, Nanomedicine in the
33
management of microbial infection - Overview and perspectives, Nano Today. 9
(2014) 479–498. doi:10.1016/j.nantod.2014.06.003.
[7] N. Tran, P.A. Tran, Nanomaterial-based treatments for medical device-associated
infections, ChemPhysChem. 13 (2012) 2481–2494.
doi:10.1002/cphc.201200091.
[8] S.M. Vetter, P.M. Schlievert, Glycerol monolaurate inhibits virulence factor
production in Bacillus anthracis, Antimicrob Agents Chemother. 49 (2005)
1302–1305.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&do
pt=Citation&list_uids=15793101.
[9] P.M. Schlievert, M.L. Peterson, Glycerol monolaurate antibacterial activity in
broth and biofilm cultures, PLoS One. 7 (2012).
doi:10.1371/journal.pone.0040350.
[10] H. Fessi, F. Puisieux, J.P. Devissaguet, N. Ammoury, S. Benita, Nanocapsule
formation by interfacial polymer deposition following solvent displacement, Int.
J. Pharm. 55 (1989) R1–R4. doi:10.1016/0378-5173(89)90281-0.
[11] CLSI, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That
Grow Aerobically, 2012.
[12] I. Sondi, B. Salopek-Sondi, Silver nanoparticles as antimicrobial agent: A case
study on E. coli as a model for Gram-negative bacteria, J. Colloid Interface Sci.
275 (2004) 177–182. doi:10.1016/j.jcis.2004.02.012.
[13] M.E. Sandberg, D. Schellmann, G. Brunhofer, T. Erker, I. Busygin, R. Leino, et
al., Pros and cons of using resazurin staining for quantification of viable
Staphylococcus aureus biofilms in a screening assay, J. Microbiol. Methods. 78
(2009) 104–106. doi:10.1016/j.mimet.2009.04.014.
[14] M. Sandberg, A. Määttänen, J. Peltonen, P.M. Vuorela, A. Fallarero, Automating
a 96-well microtitre plate model for Staphylococcus aureus biofilms: an approach
to screening of natural antimicrobial compounds, Int. J. Antimicrob. Agents. 32
(2008) 233–240. doi:10.1016/j.ijantimicag.2008.04.022.
[15] S. Manner, M. Skogman, D. Goeres, P. Vuorela, A. Fallarero, Systematic
exploration of natural and synthetic flavonoids for the inhibition of
Staphylococcus aureus biofilms, Int. J. Mol. Sci. 14 (2013) 19434–19451.
doi:10.3390/ijms141019434.
[16] E. Turos, J.Y. Shim, Y. Wang, K. Greenhalgh, G.S.K. Reddy, S. Dickey, et al.,
Antibiotic-conjugated polyacrylate nanoparticles: New opportunities for
development of anti-MRSA agents, Bioorganic Med. Chem. Lett. 17 (2007) 53–
56. doi:10.1016/j.bmcl.2006.09.098.
34
[17] A. Baelo, R. Levato, E. Julián, A. Crespo, J. Astola, J. Gavaldà, et al.,
Disassembling bacterial extracellular matrix with DNase-coated nanoparticles to
enhance antibiotic delivery in biofilm infections, J. Control. Release. 209 (2015)
150–158. doi:10.1016/j.jconrel.2015.04.028.
[18] V.M. Comin, L.Q.S. Lopes, P.M. Quatrin, M.E. de Souza, P.C. Bonez, F.G.
Pintos, et al., Influence of Melaleuca alternifolia oil nanoparticles on aspects of
Pseudomonas aeruginosa biofilm, Microb. Pathog. 93 (2016) 120–125.
doi:10.1016/j.micpath.2016.01.019.
[19] N. Moghadas-Sharif, B.S. Fazly Bazzaz, B. Khameneh, B. Malaekeh-Nikouei,
The effect of nanoliposomal formulations on Staphylococcus epidermidis
biofilm., Drug Dev. Ind. Pharm. 9045 (2014) 1–6.
doi:10.3109/03639045.2013.877483.
[20] B. Khameneh, M. Iranshahy, M. Ghandadi, D. Ghoochi Atashbeyk, B.S. Fazly
Bazzaz, M. Iranshahi, Investigation of the antibacterial activity and efflux pump
inhibitory effect of co-loaded piperine and gentamicin nanoliposomes in
methicillin-resistant Staphylococcus aureus., Drug Dev. Ind. Pharm. 9045 (2014)
1–6. doi:10.3109/03639045.2014.920025.
[21] R.P. Raffin, E.S. Obach, G. Mezzalira, A.R. Pohlmann, S.S. Guterres,
Nanocápsulas poliméricas secas contendo indometacina: Estudo de formulação e
de tolerância gastrintestinal em ratos, Acta Farm. Bonaer. 22 (2003) 163–172.
[22] C.E. Mora-Huertas, H. Fessi, A. Elaissari, Polymer-based nanocapsules for drug
delivery, Int. J. Pharm. 385 (2010) 113–142. doi:10.1016/j.ijpharm.2009.10.018.
[23] S.R. Schaffazick, S.S. Guterres, L. De Lucca Freitas, A.R. Pohlmann,
Caracterização e estabilidade físico-química de sistemas poliméricos
nanoparticulados para administração de fármacos, Quim. Nova. 26 (2003) 726–
737. doi:10.1590/S0100-40422003000500017.
[24] M. Instruments, Zeta potential: An Introduction in 30 minutes, Zetasizer Nano
Serles Tech. Note. MRK654-01. 2 (2011) 1–6.
http://scholar.google.com/scholar?hl=en&btnG=Search&q=intitle:Zeta+Potential
+An+Introduction+in+30+Minutes#0.
[25] R.P. Raffin, L.M. Colomé, S.E. Haas, D.S. Jornada, A.R. Pohlmann, S.S.
Guterres, Development of HPMC and Eudragit S100® blended microparticles
containing sodium pantoprazole, Pharmazie. 62 (2007) 361–364.
doi:10.1691/ph.2007.5.6077.
[26] K.P. Seremeta, D.A. Chiappetta, A. Sosnik, Poly(e{open}-caprolactone),
Eudragit?? RS 100 and poly(e{open}-caprolactone)/Eudragit?? RS 100 blend
submicron particles for the sustained release of the antiretroviral efavirenz,
35
Colloids Surfaces B Biointerfaces. 102 (2013) 441–449.
doi:10.1016/j.colsurfb.2012.06.038.
[27] A. De Arce Velasquez, L.M. Ferreira, M.F.L. Stangarlin, C.D.B. Da Silva,
C.M.B. Rolim, L. Cruz, Novel Pullulan-Eudragit?? S100 blend microparticles for
oral delivery of risedronate: Formulation, in vitro evaluation and tableting of
blend microparticles, Mater. Sci. Eng. C. 38 (2014) 212–217.
doi:10.1016/j.msec.2014.02.003.
[28] L.Q.S. Lopes, C.G. Santos, R.D.A. Vaucher, R.P. Raffin, R.C. V Santos,
Evaluation of antimicrobial activity of glycerol monolaurate nanocapsules
against american foulbrood disease agent and toxicity on bees, Microb. Pathog.
(2016). doi:10.1016/j.micpath.2016.05.014.
[29] F. Esmaeili, M. Hosseini-Nasr, M. Rad-Malekshahi, N. Samadi, F. Atyabi, R.
Dinarvand, Preparation and antibacterial activity evaluation of rifampicin-loaded
poly lactide-co-glycolide nanoparticles, Nanomedicine Nanotechnology, Biol.
Med. 3 (2007) 161–167. doi:10.1016/j.nano.2007.03.003.
[30] H. sheng Peng, X. jun Liu, G. xiang Lv, B. Sun, Q. fei Kong, D. xu Zhai, et al.,
Voriconazole into PLGA nanoparticles: Improving agglomeration and antifungal
efficacy, Int. J. Pharm. 352 (2008) 29–35. doi:10.1016/j.ijpharm.2007.10.009.
[31] V.C.F. Mosqueira, P. Legrand, A. Gulik, O. Bourdon, R. Gref, D. Labarre, et al.,
Relationship between complement activation, cellular uptake and surface
physicochemical aspects of novel PEG-modified nanocapsules, Biomaterials. 22
(2001) 2967–2979. doi:10.1016/S0142-9612(01)00043-6.
36
Legends
Table 1. MIC and MBC of GML and GML nanocapsules against C. albicans.
Fig. 1. GML nanocapsule in TEM
Fig. 2. Growth curve dependent of dose of GML and GML nanocapsules against C.
albicans.
Fig. 3. Biofilm quantification by Crystal violet (A) and safranin (B) stain after exposure GML
or GML Nanocapsules. Was used analysis of variance (ANOVA) followed by Tukey test
considering values p < 0.05 statistically significant comparing with Positive Control. Data
expressed on Average ± Standard derivation. Absorbance at 570 nm.
Fig. 4. Well-plate semi-quantification of biofilm by crystal violet (A to C) and safranin (D to F).
Positive control (A,D), GML (B,E) and GML nanocapsules (C,F).
Fig. 5. Efficiency of biofilm inhibition of GML and GML Nanocapsules on many
concetrations.
Fig. 6. Kinetics of inhibition of formed C. albicans biofilm with GML and GML
Nanocapsules.
Fig. 7. Biofilm stained with Calcofluor White. Positive Control (a), GML (b) and GML
nanocapsules (c).
37
Fig. 1. GML nanocapsule in TEM
38
Fig. 2. Growth curve dependent of dose of GML and GML nanocapsules against C.
albicans.
0.0 31.2 62.40.0
0.1
0.2
0.3
0.4
0.5Positive Control
GML
GML nanocapsules
7.8 15.6
Concentration (g/mL)
Ab
orb
an
ce (
OD
60
0)
39
Fig. 3. Biofilm quantification by Crystal violet (A) and safranin (B) stain after exposure
GML or GML Nanocapsules. Was used analysis of variance (ANOVA) followed by
Tukey test considering values p < 0.05 statistically significant comparing with Positive
Control. Data expressed on Average ± Standard deviation and absorbance reader with
wavelength at 570 nm.
40
Fig. 4. Well-plate semi-quantification of biofilm by crystal violet (A to C) and safranin
(D to F). Positive control (A,D), GML (B,E) and GML nanocapsules (C,F).
41
Fig. 5. Efficiency of biofilm inhibition of GML and GML Nanocapsules on many
concetrations. Was used analysis of variance (ANOVA) followed by Tukey test
considering values p < 0.001 (***) statistically significant comparing with Control.
42
Fig. 6. Kinetics of inhibition of formed C. albicans biofilm with GML and GML
Nanocapsules.
0 24 480
500
1000
1500 Positive Control
GML Nanocapsules
GML
3 6 12
Time (h)
Ab
so
rban
ce (
OD
54
0)
43
Fig. 7. Biofilm stained with Calcofluor White. Positive Control (A), GML (B) and
GML Nanocapsules (C).
44
Table 1. MIC and MBC of GML and GML nanocapsules against C. albicans.
MIC (µg/mL) MBC (µg/mL)
Microorganism GML GML
nanocapsules
GML GML
nanocapsules
C. albicans ATCC 14053 31.25 15.5 62.5 31.25
45
3.1.2 ARTIGO 2: CHARACTERISATION AND ANTI-BIOFILM ACTIVITY
OF GLYCEROL MONOLAURATE NANOCAPSULES AGAINST
PSEUDOMONAS AERUGINOSA
O segundo trabalho teve como objetivo avaliar o potencial antibiofilme das
nanocápsulas contra o bacilo Gram negativo Pseudomonas aeruginosa. O artigo conta
com:
Produção e caracterização da formulação contendo as nanocápsulas;
Determinação da concentração inibitória e bactericida mínima;
Realização da curva de crescimento microbiano;
Atividade antibiofilme pela quantificação da biomassa, proteínas e
polissacarídeos;
Capacidade de inibir a formação do biofilme pelo MLG e as nanocápsulas;
Microscopia de Força Atômica dos biofilmes formados em poliestireno;
46
Characterisation and anti-biofilm activity of glycerol monolaurate nanocapsules
against Pseudomonas aeruginosa
Leonardo Quintana Soares Lopesa,b*
, Rodrigo de Almeida Vaucher c
, Janice Luehring
Giongo d , André Gündel
e, Roberto Christ Vianna Santos
a,b
a Post Graduate Program in Nanosciences, Universidade Franciscana, Santa Maria,
Brazil
b Microbiology and Parasitology Department, Health Sciences Center, Universidade
Federal de Santa Maria, Santa Maria, Brazil
c Laboratory of Research in Biochemistry and Molecular Biology of Microorganisms,
Post graduate Program in Biochemistry and Bioprospecting, Universidade Federal de
Pelotas, Capão do Leão, Brazil
d Pharmacy Department, Faculdade Anhanguera, Pelotas, Brazil
e Universidade Federal do Pampa, Bagé, Brazil
#Correspondent author e-mail address: [email protected]
Permanent address: Universidade Franciscana, Laboratory of Microbiology Reserach
Rua dos Andradas 1614, Santa Maria-RS, Zip Code 97010-032, Brazil
mailto:[email protected]
47
Abstract
Pseudomonas aeruginosa is a ubiquitous microorganism that commonly causes
hospital-acquired infections, including pneumonia, bloodstream and urinary tract
infections and it is well known for chronically colonising the respiratory tract of patients
with cystic fibrosis, causing severe intermittent exacerbation of the condition. P.
aeruginosa may appear in the free form cell but also grows in biofilm communities
adhered to a surface. An alternative to conventional antimicrobial agents are
nanoparticles that can act as carriers for antibiotics and other drugs. In this context, the
study aimed to characterise and verify the anti-biofilm potential of GML Nanocapsules
against P. aeruginosa. The nanocapsules showed a mean diameter of 190.7 nm,
polydispersion index of 0.069, the zeta potential of -23.3 mV. The microdilution test
showed a MIC of 62.5 µg/mL to GML and 15.62 µg/mL to GML Nanocapsules. The
anti-biofilm experiments demonstrated the significant reduction of biomass, proteins,
polysaccharide and viable P. aeruginosa in biofilm treated with GML Nanocapsules
while the free GML did not cause an effect. The AFM images showed a decrease in a
biofilm which received GML. The positive results suggest an alternative for the public
health trouble related to infections associated with biofilm.
Keywords: Biofilm; P. aeruginosa; Nanocapsules; Glycerol monolaurate, Atomic
Force Microscopy;
48
1. Introduction
Pseudomonas aeruginosa is a pathogen that is commonly responsible for acute and
chronic respiratory infections, associated with a high mortality [1]. The emergence of
pan-resistant strains such as P. aeruginosa and the ability of many pathogens to form
multidrug-resistant biofilms during infection increases the threat of bacterial diseases
that are untreatable with current antibiotics [2]. A biofilm is a matrix-enclosed bacterial
population in which bacteria adhere both to surfaces or interfaces [3,4]. The matrix
where microorganisms are found usually is composed of an extracellular polymeric
substance containing proteins, polysaccharides, lipids and extracellular DNA [5,6].
Biofilms are typically resistant to therapeutic concentrations of antibiotics that are based
in part on the MICs of planktonic cells [7–9].
Glycerol monolaurate (GML) is a mild surfactant formed by glycerol and lauric acid. It
is used in the cosmetic and food industry as a preservative and emulsifier also is
generally recognised as safe (GRAS) for oral use by the FDA [10]. GML has
antimicrobial activity against enveloped viruses [11] and a variety of bacteria, including
some Gram-negative bacteria such as Gardnerella vaginalis [12] and some Gram-
positive bacteria such as Streptococcus spp. [13] and Staphylococcus aureus [14,15].
However, the use of GML is not expanded due to its low solubility in water, leading to a
low bioavailability [16].
In view of the grave healthcare concern associated with bacterial biofilms, new
approaches to biofilm growth inhibition, biofilm disruption or biofilm eradication have
been studied [17–19]. It is essential to improve the penetrative capabilities of existing
antimicrobial agents in order to overcome biofilm barriers and to achieve total
elimination of biofilms [20]. The utility of nanomaterials for efficient delivery of
49
antibacterial and development of anti-biofilm agents is well documented [21–25]. In
this context, drug delivery by polymeric nanoparticles is considered a promising
strategy to overcome the resistance of biofilms of P. aeruginosa [26,27] due to the fact
that the nanoencapsulation of some compounds represents an increase of solubility,
potential antimicrobial and consequently a decrease of toxicity [28]. In view of this, the
aims of the study are to produce and characterise GML nanocapsules and evaluate the
effectiveness to combat the Pseudomonas aeruginosa biofilms.
2. Materials and methods
2.1.Development of GML nanocapsules
The nanocapsules (GML Nanocapsules) were prepared according to the method
described previously [29] with modifications. The aqueous phase was prepared with
polysorbate 80 (0.194 g) and purified water (134 mL) at 40°C under moderated stirring
(1000 rpm). In the organic phase, the GML (0.25 g) was solubilised with sorbitan
monooleate (0.194 g), capric/caprylic triglyceride (0.8 g), and polymeric blende
PMMA-PEG (0.25 g) in acetone (67 mL) at 40°C under moderated magnetic stirring
(1000 rpm). After solubilisation, the organic phase was poured into the aqueous phase
under magnetic stirring which was maintained for 10 min. The organic solvent and the
water were evaporated in a rotary evaporator at 40°C to adjust the concentration to 1
mg/mL getting 25 mL of formulation. A blank formulation (Blank Nanocapsules) was
developed in the same way as GML Nanocapsules (but without GML).
2.2.Nanocapsule characterisation
The formulations were characterised as diameter distribution and polydispersion index
(PDI) by dynamic light scattering (DLS), diluting (500×) the nanocapsules in Milli-Q®
50
water. The zeta potential was evaluated by electrophoresis in a Zetasizer Nano-ZS
(Malvern Instruments, United Kingdom), diluting (500×) the formulation in 1 mM
NaCl. The pH was evaluated using a potentiometer (Digimed®). Each parameter was
evaluated in triplicate (n=3) and results were expressed by the mean ± standard
deviation (SD).
2.3.Microorganism
The strain Pseudomonas aeruginosa PAO1 was obtained by the American Type Culture
Collection. This microorganism was maintained on the culture medium with glycerol
and cooled at -80°C. The sample was unfrozen, inoculated on Brain Heart Infusion
broth (BHI) and incubated for 24 hours. After, it was seeded on Nutrient agar and
incubated for 24 hours at 37°C.
2.4.Minimal Inhibitory Concentration (MIC) and Minimal bactericidal
concentration (MBC)
The MIC was performed by microdilution method on 96-well plate [30]. Different
concentrations GML and GML Nanocapsules were add-on wells containing Mueller
Hinton broth (MHB). The positive control was considered the well with inoculum in
MHB and negative control only MHB with saline. The assay was performed in five
replicates. After the process, the plate was incubated for 24 hours at 37°C. After
incubation, it was added to 100 µL of 0.2% 2,3,5 triphenyl tetrazolium chloride (TTC).
The lower concentration that did not show visible microbial growth (reddish colour due
to the presence of TTC) was considered the minimum inhibitory concentration. To
determine the MBC, an aliquot of 1 µL was taken of each well, seeded on Nutrient agar
51
plate and incubated to 24 hours. After the colonies were identified and the lowest
concentration which does not demonstrate microbial growth was considered the MBC.
2.5.Biofilm formation and treatment
The biofilm was formed according to the conditions previously optimised and described
[31,32] with modifications. The fresh, exponentially grown culture of P. aeruginosa
was diluted to be 106 CFU/mL and 15 µL was added to flat-bottomed 96-well plates
(Nunclon™ D surface, Nunc, Roskilde, Denmark), containing 100 µL of BHI broth and
the plate was incubated in 37°C, for 24 hours. After the formation of biofilm, it was
added 100 µL of GML or GML nanocapsules solution. The GML in the concentration
of 62.5 and 125 µg/mL while the GML nanocapsules in the concentration of 15.62 and
62.5 µg/mL corresponding to MIC and MBC. After the addition, the treatment plate was
incubated for 24 hours in the condition of 37°C according to Manner et al. [33]. The
positive control was performed containing only BHI broth and the P. aeruginosa strain
while the negative control was only BHI broth.
2.6.Quantification of biofilm biomass
After the treatment, the supernatant was removed and washed three times with distilled
water and then, the quantifications were performed. The treated biofilm was fixed with
95% of methanol and stained with 150 µL of 0.1% of crystal violet 15 min at room
temperature (RT). After incubation, the well-plates were washed with distilled water.
Ethanol 95% was added to dissolve the colouring and after, 100 µL transferred to
another plate to measure spectrophotometrically at 540 nm in a spectrophotometer (TP-
Reader; ThermoPlate, Goiás, Brazil). The biofilm formation was determined by the
52
difference between the mean OD readings obtained in the positive control (BHI broth
and P. aeruginosa strain) and the treatment with GML and GML nanocapsules.
2.7.Quantification of Biofilm Cultivable Cells
The number of cultivable biofilm cells was determined by counting colony forming
units (CFUs) following biofilm cells suspension. Briefly, biofilms were first washed
twice in PBS to remove loosely attached cells and the biofilm was then suspended in
PBS by repeated pipetting. Complete removal of the biofilm was confirmed by
subsequent crystal violet staining and spectrophotometric reading for inspection of the
wells. The suspended biofilm (100 µL in PBS) was vigorously vortexed for 5 min to
disrupt the biofilm matrix and serial decimal dilutions (in PBS) were plated onto
Nutrient. Agar plates were incubated for 24 h at 37°C and the colony-forming units per
millilitre (CFU/mL) were counted [34]. Experiments were repeated in three independent
experiments in five replicates.
2.8.Determination of biofilm polysaccharide levels
Polysaccharides into the biofilm were measured by the Phenol-sulfuric acid method
[35]. After the biofilm formation and treatment, the well-plates were rinsed with PBS to
remove medium and non-adherent cells. It was added to 40 µL of deionised water, 40
µL of 5% phenol solution and 200 µL of 95-97% sulfuric acid. The plate was incubated
for 30 minutes at room temperature. The absorbance was read at 490 nm with a
microplate reader. Different concentrations of glucose were used as standard values for
the conversion of absorbance to polysaccharide concentrations.
53
2.9.Determination of biofilm protein levels
Protein was measured by the Bradford method of protein determination in biological
samples [36]. Briefly, a culture media with non-adherent cells was removed from wells
and the plate was washed with PBS. NaOH 0.2N was added to each well and the plate
was sonicated for 3 s. The Bradford reagent was added to each well and the plate was
incubated for 5 minutes. The absorbance was read at 595 nm with a microplate reader.
Different concentrations of bovine serum albumin were used as standard values for the
conversion of optical density to protein concentrations.
2.10. Effect against biofilm formation
The GML and GML nanocapsules were tested to verify the ability to prevent the
biofilm formation of P. aeruginosa. The inoculum containing the microorganism was
added into the well-plate with BHI broth and sub inhibitory concentrations (0.5xMIC or
0.25xMIC) of GML and GML nanocapsules. The plate was incubated at 37°C for 24
hours. The biofilm was fixed with 95% of methanol and stained with 150 µL of 0.1% of
crystal violet 15 min at RT. The well-plates were washed with distilled water and
Ethanol 95% was added to dissolve the colouring. After this, 100 µL was transferred to
another plate to measure in a spectrophotometer (TP-Reader; ThermoPlate, Goiás,
Brazil) at 540 nm. Only BHI broth with P. aeruginosa was considered a positive control
and the percentage of inhibition was calculated by OD Test/OD Control × 100.
2.11. Growth curve analysis
The antimicrobial activity of GML and GML nanocapsules was evaluated by growth
curve analysis. Overnight culture of PAO1 (1%; 0.4 OD at 600 nm) was inoculated in a
96-well plate with 100 µL of Mueller Hinton broth supplemented with GML (MBC –
54
125 µg/mL) or GML Nanocapsules (MBC – 62.5 µg/mL). The plate was incubated at
37°C and the cell density was measured by microplate reader at intervals of 0, 6, 12, 24
and 48 h.
2.12. Atomic Force Microscopy (AFM)
After the treatment (GML and GML Nano), the well plate was cut and fixed with
absolute methanol for 1 minute (the well without treatment was considered as Control).
The images were obtained using Agilent Technologies 5500 microscopy. The images
(10 μm × 10 μm) were collected in a non-contact mode using PPP-NCL tips
(Nanosensors, force constant = 48 N/m). The images were analysed using PicoView
software.
2.13. Statistical analysis
The results of the microbiological assays were submitted to One-way analysis of
variance (ANOVA) following by Tukey test with 95-99% of significance. The
experiments were performed in five replicates and three independent experiments.
3. Results
Nanocapsules characterization
The formulation was evaluated as physicochemical parameters. The measurements of
GML nanocapsules showed a mean diameter of 190.7 ± 2, polydispersion index of
0.069 ± 0.013, zeta potential of -23.3 ± 3 and pH of 6.11 ± 0.18. The blank
nanocapsules demonstrate a mean diameter of 176.5 ± 4, polydispersion index of 0.042
± 0.025, zeta potential of -12.8 ± 6 and pH of 6.22 ± 0.11. The graph of the light
scattering measurements showing the size distribution is demonstrated in Figure 1.
55
Minimal Inhibitory Concentration (MIC) and Minimal bactericidal concentration
(MBC)
The MIC and MBC can be visualised in Table 1. The Blank Nanocapsules was tested in
the same way with GML Nanocapsules but without antimicrobial activity (data not
shown). The MIC and MBC of GML Nanocapsules were 25 and 50% less compared to
free GML.
Quantification of biofilm biomass
After colouring, the absorbance was read. The results showed a decrease in 78%
approximately of biofilm biomass treated with GML Nano, while the GML reduced by
57% (Figure 2).
Quantification of Biofilm Cultivable Cells
The biofilm was suspended in NaCl and seeded into Nutrient agar. The plate was
incubated and the colonies were counted. The experiment was performed in five
replicates in two independents experiments. The result of counting was shown in Figure
3.
Determination of biofilm polysaccharide levels
The polysaccharides of biofilms treated with GML and GML Nano were quantified.
The assay demonstrated the reduction of polysaccharides in 58 and 61% treated with
GML and GML Nano respectively. The results are shown in Figure 4.
Determination of biofilm protein levels
56
After the protein measurement, the experiment showed an increase of proteins levels on
biofilm treated with free GML, while the GML Nano caused a reduction of 59% in
proteins (Figure 5).
Inhibition of biofilm
The assay demonstrated that the GML did not significantly inhibit biofilm formation
while the GML Nano inhibited by about 37% in a sub-inhibitory concentration. The
results are shown in Figure 6.
Growth curve analysis
The concentration used in this test was 125 µg/mL for GML and 62.5 µg/mL of GML
nanocapsules. After the absorbance reader, the result was showed in Figure 7. The assay
demonstrates that the treatment with free GML has no effect on the number of
microorganisms. The treatment with GML Nanocapsules showed a significant decrease
in comparison with the Control and free GML.
Atomic Force Microscopy
Figure 8 shows the AFM results of the polystyrene not treated (Control) and treated
with free GML and GML Nanocapsules for P. aeruginosa PAO1 strain. The images
show high peaks in the control (6 µm) indicating the formation of a biofilm. In the
sample treated with the free GML a slight decrease (4 µm) in the biofilm while the
sample treated with GML Nanocapsules demonstrated a great effectiveness in the action
against formed biofilm with peaks up to 0.8 µm.
57
4. Discussion
The importance of these results lies in the fact that these microorganisms isolated from
the hospital environment can colonise and adhere to surfaces of medical instruments and
implants. In addition, because they are already resistant to multiple drugs, the adhesion
capability can effectively reduce the antimicrobial options and further aggravate the
infectious condition.
The results of the characterisation of nanoparticles indicates an adequate homogeneity,
all formulations must be monodisperse (PDI < 0.25) and a diameter smaller than 300
nm. Moreover, negative values to zeta potential are expected when used polymers
containing a grouping ester in structure, such as PMMA [37]. When the nanostructure
shows an elevated charge in modulus, the system stability tends to be superior with a
lower probability for particle aggregation [38,39]. The obtained results of
characterisation in the present study corroborate with works related in the literature
which use a polymeric blend in the development of nanoparticles such carries and
suggest homogeneity on size distribution [40–42].
The ability of P. aeruginosa PAO1 to form biofilm is one of the causes that make an
important pathogen. The resistance to antibiotic therapy and the morbidly associated
with this bacteria are attributed factors to your transition in host tissues (mainly lung,
skin and bladder) of planktonic forms to biofilm style [43,44]. The appearance of multi-
resistant strains is worrying, being necessary for the appearance of new therapeutic
approaches [45].
In our work used the gram-negative P. aeruginosa PAO1, which have an EPS
composed mainly of polysaccharides, protein, nucleic acids, lipids and humic
58
substances. The gram-negative bacteria have a negative charge on your surface,
originated from lipopolysaccharides and proteins of the outside membrane.
A recent study of our group with GML Nanocapsules showed the antimicrobial activity
against bee pathogens [16]. This is the first report which shows GML nanoparticles
against Pseudomonas aeruginosa biofilms. The assay with crystal violet demonstrated
the high antibiofilm activity of GML Nanocapsules, while the GML doesn’t show
significant effect being equal to the positive control (Figure 2). Studies performed by
Schlievert et al. [13] demonstrated the capacity of GML to inhibiting Staphylococcus
aureus biofilms. In the present study the GML Nanocapsules demonstrated the ability to
prevent biofilm formation (Fig. 6).
Moreover, the slow release of the GML could have had an important role in anti-biofilm
activity throughout the time of the assay. In addition, a long-time of release could help
the dispersion of GML increasing cellular penetration [46].
After the antimicrobial tests, it was possible to observe that the GML in free form did
not decrease the microbial population in 48h, while the GML Nanocapsules almost
completely reduced the microorganisms. The positive control (containing the only
microorganism) did not present a fall in the number of bacteria. The experiment showed
the controlled release of the compound, the characteristic of the nanostructured system
[47,48].
The structure of biofil
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