Estratégia com tt-farnesol, miricetina e composto 1771 associados à nanopartícula carreadora é eficaz na prevenção de biofilmes cariogênicos formados por Streptococcus mutans e Candida albicans in vitro
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Data
2021-11-08
Autores
Rocha, Guilherme Roncari
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O desenvolvimento de terapias eficazes para o controle de biofilme oral é um desafio. Agentes tópicos usados para tratar e/ou prevenir doenças orais causadas por biofilme, geralmente, não são mantidos na boca por um período suficiente para que os mesmos possam exercer seu potencial terapêutico máximo. Assim, foram desenvolvidas nanopartículas carreadoras (NPC) de fármaco que se aderem à hidroxiapatita, à película salivar e aos exopolissacarídeos. As NPC possuem elementos pH sensíveis para a liberação controlada de fármacos em nichos acídicos de biofilmes patogênicos. O tt-farnesol e miricetina podem ser carreados e liberados com sucesso pelas NPC, diminuindo a biomassa de biofilme de Streptococcus mutans. O composto 1771 é um novo fármaco para o controle da síntese de ácidos lipoteicóicos - estruturas da parede celular de espécies Gram-positivas - responsáveis por co-agregação microbiana. O biofilme de S. mutans e Candida albicans é extremamente patogênico levando ao desenvolvimento de lesões cariosas severas em um curto período in vivo. Portanto, o objetivo foi investigar se o composto 1771 consegue ser carreado pela NPC e também o tratamento contendo a associação dos três fármacos (NPC-farnesol-miricetina-1771) consegue prevenir a formação do biofilme cariogênico mono-espécie formado por S. mutans com superfície tratada desde o início da formação (regime de prevenção) e multi-espécie formado por S. mutans e C. albicans sob o regime de prevenção e o biofilme tratado posteriormente a sua formação sobre a superfície (regime de controle) in vitro. Assim, foi necessário sintetizar uma nanopartícula e conferir sua interação com o composto 1771 e caracterizá-la, testando, posteriormente as soluções contra biofilmes cariogênicos. Os testes de caracterização da nanopartícula e da associação da nanopartícula com 1771 incluíram tamanho, carga (potencial zeta), absorbância, fluorescência, cromatografia de permeação em gel e ressonância magnética nuclear. O polímero criado foi formado por dois blocos sendo o bloco 1 (corona) responsável aderir aos exopolissacarídeos e película salivar por ser catiônica e carrear os fármacos: miricetina e composto 1771. O bloco 2 (core) é responsável por carrear fármacos insolúveis como o tt-farnesol, além de ser sensível ao pH, liberando-os principalmente com a queda do pH. Os testes de absorbância e fluorescência confirmaram a forte interação entre NPC com o composto 1771. Os demais testes caracterizaram a carga de superfície da NPC e tamanho de +23 mV e 48,3 d.nm para +32 mV e 48,3 para 75 d.nm pré/pós adição dos fármacos, respectivamente. Os testes microbiológicos foram: concentração antimicrobiana mínima usando culturas planctônicas e foram preparados dois regimes de tratamentos utilizando biofilmes formados sobre discos de hidroxiapatita com película salivar. Foi acompanhado o pH do meio de cultura, biomassa dos biofilmes e população. Foram realizadas microscopias confocal e eletrônica de varredura para caracterizar a estrutura tridimensional dos biofilmes. Menores concentrações dos fármacos carreados inibiram o crescimento bacteriano planctônico, mas não inibiram o crescimento do fungo e a NPC vazia não afetou ambos os microrganismos. A associação dos fármacos carreados previne a formação de biofilmes ao inibir a queda do pH, crescimento da população microbiana e a produção de matriz extracelular, consequentemente, a biomassa. Ocorreu melhor eficácia nos tratamentos NPC-farnesol-1771 e NPC-farnesol-miricetina-1771 no regime de prevenção, mas não controlou no regime de tratamento. Portanto, a associação dos fármacos, especialmente tt-farnesol e composto 1771 associados ao sistema de carreamento preveniu a formação de biofilme multi-espécie sobre a superfície de hidroxiapatita.
The development of effective therapies for the control of oral biofilm is a challenge. Topical agents used to treat and/or prevent oral diseases caused by biofilms, generally, are not maintained in the mouth for a sufficient time to they exercise their maximum therapeutic potential. Thus, nanoparticle carries (NPC) have been developed to adhere on hydroxyapatite, pellicle, and exopolysaccharides. NPCs has pH sensitive elements for releasing drugs controlled at acidic niches of pathogenic biofilms. The tt-farnesol and myricetin can be successfully carried and released by NPCs, decreasing biomass of Streptococcus mutans biofilm. Compound 1771 is a new drug for controlling the synthesis of lipoteichoic acids - cell wall protein from Gram-positive bacteria - responsible for microbial co-aggregation. The biofilm of S. mutans and Candida albicans is extremely pathogenic, leading to the development of severe carious lesions in a short period in vivo. Therefore, the objective were investigate whether the compound 1771 can be carried by NPC and also the treatment containing the association of the three drugs (NPC-farnesol-myricetin1771) can prevent mono-species biofilm formed by S. mutans with surface treated since the beginning of formation (prevention regimen) and multi-species formed by S. mutans and C. albicans under prevention regimen and control regimen (the biofilm was treated after stablished on the surface) in vitro. Thus, it was necessary to synthesize a nanoparticle and check the interaction with compound 1771 and characterize it, later testing the solutions against cariogenic biofilms. The tests for NPC-1771 characterization included size, zeta potential, absorbance, fluorescence, gel permeation chromatography and nuclear magnetic resonance. The polymer created was formed by two blocks, block 1 (corona) being responsible for adhering to exopolysaccharides and pellicle for being cationic nanoparticle and carrying the drugs: myricetin and compound 1771; and block 2 (core) is responsible for carrying insoluble drugs such as tt-farnesol, in addition to being pH sensitive, releasing them mainly as the pH drops. Absorbance and fluorescence tests confirmed the strong interaction between NPC and compound 1771. The other tests characterized the NPC's zeta-potential and size from +23 mV to 32 mV and from 48.3 to 75 d.nm pre/post drug association, respectively. The microbiological tests were made using minimum antimicrobial concentration using planktonic cultures. Two treatment regimens were prepared using biofilms formed on hydroxyapatite discs with pellicle. The pH of the culture medium, biomass of biofilms and population was monitored. Confocal and scanning electron microscopes were performed to characterize the three-dimensional structure. Lower concentrations of the drugs carried inhibited the planktonic bacterial growth but did not inhibit the fungus growth and the NPC free did not affect both microorganisms. The association of the drugs carried prevents the formation of biofilms by inhibiting the pH drop, microbial population and the production of extracellular matrix, consequently, biomass. The best efficacy occurred in the treatments NPC-farnesol-1771 and NPC-farnesol-myricetin-1771 in the prevention regimen. No treatments control biofilm formation in the control regimen. Therefore, the association of drugs, especially tt-farnesol and compound 1771 associated to NPC prevented the biofilm formation on hydroxyapatite surface.
The development of effective therapies for the control of oral biofilm is a challenge. Topical agents used to treat and/or prevent oral diseases caused by biofilms, generally, are not maintained in the mouth for a sufficient time to they exercise their maximum therapeutic potential. Thus, nanoparticle carries (NPC) have been developed to adhere on hydroxyapatite, pellicle, and exopolysaccharides. NPCs has pH sensitive elements for releasing drugs controlled at acidic niches of pathogenic biofilms. The tt-farnesol and myricetin can be successfully carried and released by NPCs, decreasing biomass of Streptococcus mutans biofilm. Compound 1771 is a new drug for controlling the synthesis of lipoteichoic acids - cell wall protein from Gram-positive bacteria - responsible for microbial co-aggregation. The biofilm of S. mutans and Candida albicans is extremely pathogenic, leading to the development of severe carious lesions in a short period in vivo. Therefore, the objective were investigate whether the compound 1771 can be carried by NPC and also the treatment containing the association of the three drugs (NPC-farnesol-myricetin1771) can prevent mono-species biofilm formed by S. mutans with surface treated since the beginning of formation (prevention regimen) and multi-species formed by S. mutans and C. albicans under prevention regimen and control regimen (the biofilm was treated after stablished on the surface) in vitro. Thus, it was necessary to synthesize a nanoparticle and check the interaction with compound 1771 and characterize it, later testing the solutions against cariogenic biofilms. The tests for NPC-1771 characterization included size, zeta potential, absorbance, fluorescence, gel permeation chromatography and nuclear magnetic resonance. The polymer created was formed by two blocks, block 1 (corona) being responsible for adhering to exopolysaccharides and pellicle for being cationic nanoparticle and carrying the drugs: myricetin and compound 1771; and block 2 (core) is responsible for carrying insoluble drugs such as tt-farnesol, in addition to being pH sensitive, releasing them mainly as the pH drops. Absorbance and fluorescence tests confirmed the strong interaction between NPC and compound 1771. The other tests characterized the NPC's zeta-potential and size from +23 mV to 32 mV and from 48.3 to 75 d.nm pre/post drug association, respectively. The microbiological tests were made using minimum antimicrobial concentration using planktonic cultures. Two treatment regimens were prepared using biofilms formed on hydroxyapatite discs with pellicle. The pH of the culture medium, biomass of biofilms and population was monitored. Confocal and scanning electron microscopes were performed to characterize the three-dimensional structure. Lower concentrations of the drugs carried inhibited the planktonic bacterial growth but did not inhibit the fungus growth and the NPC free did not affect both microorganisms. The association of the drugs carried prevents the formation of biofilms by inhibiting the pH drop, microbial population and the production of extracellular matrix, consequently, biomass. The best efficacy occurred in the treatments NPC-farnesol-1771 and NPC-farnesol-myricetin-1771 in the prevention regimen. No treatments control biofilm formation in the control regimen. Therefore, the association of drugs, especially tt-farnesol and compound 1771 associated to NPC prevented the biofilm formation on hydroxyapatite surface.
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Palavras-chave
Biofilme, Nanopartículas, Microbiologia, Streptococcus mutans, Candida albicans, Biofilm, Nanoparticle, Microbiology, Streptococcus mutans, Candida albicans