Avaliação do potencial anti-Helicobacter pylori de compostos de coordenação de prata (I) incorporados ou não em sistema nanoestruturado de liberação de fármacos
Data
2020-02-18
Autores
Camargo, Bruna Almeida Furquim de
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Universidade Estadual Paulista (Unesp)
Resumo
O Helicobacter pylori é uma bactéria que coloniza o epitélio gástrico e embora esteja frequentemente associado a infecções assintomáticas, pode promover o desenvolvimento de distúrbios gástricos, como úlcera péptica, gastrite aguda e crônica, tecido linfoide associado à mucosa (Linfoma de MALT) e adenocarcinomas gástricos. Encontra-se na lista de bactérias que são cada vez mais resistentes aos fármacos e apresentam uma grande ameaça à saúde pública. Os perfis de resistência aos antimicrobianos estão relacionados à ocorrência de fatores virulência dos microrganismos, especialmente a produção de biofilme. A importância do H. pylori na área da saúde bem como os problemas enfrentados no tratamento de infecções provocadas por esse microrganismo, impulsiona a busca por novas alternativas terapêuticas que visam fornecer menores efeitos colaterais, custo e maior eficácia, pois ainda há uma falha no tratamento, devido a fatores como resistência a antibióticos, consumo de vários medicamentos ao mesmo tempo, período de terapia, além dos efeitos colaterais apresentados pelos antibióticos utilizados atualmente. A prata e seus derivados compreendem importantes propriedades antimicrobianas e possuem a capacidade de formar complexos, os quais atraem interesse ao fornecer novas abordagens medicamentosas. Assim, a incorporação em sistemas de liberação de fármacos denota-se como estratégia para aprimorar a atividade antimicrobiana de compostos metálicos, bem como possibilitar uma melhor solubilização e liberação dos mesmos. Este trabalho visou avaliar o potencial antibacteriano in vitro dos compostos [Ag(PhTSC∙HCl)2](NO3)∙H2O (1), [Ag(HTSC∙HCl)2](NO3)∙H2O (2), [Ag(PhTSC∙HCl)2](Cl)∙H2O (3) e [Ag(HTSC∙HCl)2](Cl)∙H2O (4), não incorporados e incorporados em nanopartícula polimérica (NP) frente ao H. pylori ATCC 43504. O composto que apresentou melhor atividade (não incorporado e incorporado) foi avaliado sobre a formação de biofilme e biofilme maduro do H. pylori e avaliados quanto ao potencial citotóxico sobre as linhagens celulares MRC-5 e J774A.1, potencial mutagênico e potencial tóxico em modelo alternativo in vivo de Galleria mellonella. A NP constituída de polímero poli (ɛ- caprolactona), de um tensoativo (poloxamer 407) e de uma fase aquosa (tampão fosfato pH 7,4) foi preparada por nanoprecipitação. Os ensaios de caracterização do sistema (dispersão de luz dinâmica, potencial zeta e microscopia eletrônica de transmissão (MET)) mostraram que as NPs possuem tamanhos de partículas de 155,10 nm ± 3,60 a 180,0 nm ± 1,13, índice de polidispersão de 0,093 ± 0,028 a 0,266 ± 0,032 e potencial zeta de -3,17 mV ± 0,359 a - 0,16 mV ± 0,045. Foi realizado a estabilidade das formulações por 90 dias, e diante dos resultados de CIM, juntamente com a estabilidade, foi selecionando a nanopartícula incorporada com o composto 1 (NP1) como melhor amostra para seguir com os demais ensaios. As imagens de MET confirmaram a formação das NPs. A atividade antibacteriana foi verificada através da técnica de microdiluição. O composto 1 exibiu uma Concentração Inibitória Mínima (CIM) de 3,90 μg/mL e após a incorporação sua ação foi potencializada para 0,78 μg/mL e uma Concentração Bactericida Mínima (CBM) de 7,81 μg/mL livre para 1,56 μg/mL na NP1. Para o biofilme, a NP1 apresentou melhor atividade frente ao composto 1, tanto na formação do biofilme, quanto no biofilme maduro de H. pylori. A formação do biofilme foi analisada em diferentes tempos de formação (4, 24 e 36 horas) o qual foi proposto como 4 horas o tempo suficiente para o H. pylori formar o biofilme. Na análise de citotoxicidade, o sistema possibilitou melhores resultados, proporcionando um aumento de seletividade ao H. pylori de cerca de 7 vezes para MRC-5 e 3 vezes mais quando comparado a J774A.1, além da NP1 não apresentar citotoxicidade frente as linhagens celulares na mesma concentração referente ao CIM. NP1 permitiu a inibição da mutagenicidade apresentada pelo composto livre. No ensaio in vivo em Galleria mellonella, o sistema permitiu a sobrevivência das larvas na concentração máxima preparada. Está é a primeira demonstração de atividade de complexos de prata em associação a nanopartícula polimérica contra H. pylori e indica um potencial fármaco contra H. pylori
Helicobacter pylori is a bacterium that colonizes gastric epithelium and although often associated with asymptomatic infections, it can promote the development of gastric disorders such as peptic ulcer, acute and chronic gastritis, mucosa-associated lymphoid tissue (MALT lymphoma) and gastric adenocarcinomas. It is on the list of bacteria that are increasingly resistant to drugs and pose a major threat to public health. Antimicrobial resistance profiles are related to the occurrence of virulence factors of microorganisms, especially biofilm production. The importance of H. pylori in health, as well as the difficulties faced in the treatment of infections caused by this microorganism, drives the search for new therapeutic alternatives that aim to provide lower side effects, cost and greater effectiveness, because there is still a failure in treatment, due to factors such as antibiotic resistance, consumption of several drugs at the same time, period of therapy in addition to the effects caused by the antibiotics currently used. Silver and its derivatives have important antimicrobial properties and have the ability to form complexes, which attract interest in providing new drug approaches. Thus, the incorporation in drug release systems is presented as a strategy to improve the antimicrobial activity of metallic compounds, as well as to enable a better solubilization and release of them. This work aimed to evaluate the activity of silver (I) coordination compounds: [Ag(PhTSC∙HCl)2](NO3)∙H2O (1), [Ag(HTSC∙HCl)2](NO3)∙H2O (2), [Ag(PhTSC∙HCl)2](Cl)∙H2O (3) and [Ag(HTSC∙HCl)2](Cl)∙H2O (4), unloaded and loaded into a polymeric nanoparticle (PN) against H. pylori ATCC 43504. The compound that showed the best activity (unloaded and loaded) was evaluated on the formation of mature H. pylori biofilm and biofilm and evaluated for cytotoxic potential on MRC- 5 and J774A.1 cell line, mutagenic potential and toxic potential in alternative in vivo model of Galleria mellonella. The PN composed of polymer (polycaprolactone), a surfactant (poloxamer 407) and an aqueous phase (phosphate buffer pH 7.4) was prepared by nanoprecipitation. The system characterization assays (dynamic light scattering, zeta potential and transmission electron microscopy (TEM)) showed that the PNs have particle sizes from 155.10 nm ± 3.60 to 180.0 nm ± 1.13, polydispersity index from 0.093 ± 0.028 to 0.266 ± 0.032 and zeta potential from -3.17 mV ± 0.359 to - 0.16 mV ± 0.045. The stability of the formulations was performed for 90 days, and in view of the MIC resultd, together with the stability, the nanoparticle incoporated with compound 1 (PN1) was selected as the best sample to follow with the other tests. TEM images confirmed the formation of PNs. Antibacterial activity was evaluated by the microdilution technique. The unloaded compound 1 had a Minimum Inhibitory Concentration (MIC) of 3.90 μg/mL and after loaded its action was potentiated to 0.78 μg/mL and a Minimum Bactericidal Concentration (MBC) of 7.81 μg/mL free for 1.56 μg/mL in PN1. For biofilm, PN1 showed better activity against compound 1, both in biofilm formation and in mature H. pylori biofilm. Biofilm formation was analyzed at different formation times (4, 24 and 36 hours) which was proposed as 4 h long enough for H. pylori to form the biofilm. In the cytotoxicity analysis, the system provided better results, providing H. pylori selectivity increase of about 7 times for MRC-5 and 3 times more when compared to J774A.1, in addition to PN1 not showing cytotoxicity against cell lines at the same concentration as MIC. PN1 allowed the inhibition of the mutagenicity presented by the free compound. In the in vivo assay on Galleria mellonella, the system allowed larvae survival at the maximum concentration prepared. This is the first demonstration of the activity of silver complexes in association with a polymeric nanoparticle against H. pylori and indicates a potential drug against H. pylori.
Helicobacter pylori is a bacterium that colonizes gastric epithelium and although often associated with asymptomatic infections, it can promote the development of gastric disorders such as peptic ulcer, acute and chronic gastritis, mucosa-associated lymphoid tissue (MALT lymphoma) and gastric adenocarcinomas. It is on the list of bacteria that are increasingly resistant to drugs and pose a major threat to public health. Antimicrobial resistance profiles are related to the occurrence of virulence factors of microorganisms, especially biofilm production. The importance of H. pylori in health, as well as the difficulties faced in the treatment of infections caused by this microorganism, drives the search for new therapeutic alternatives that aim to provide lower side effects, cost and greater effectiveness, because there is still a failure in treatment, due to factors such as antibiotic resistance, consumption of several drugs at the same time, period of therapy in addition to the effects caused by the antibiotics currently used. Silver and its derivatives have important antimicrobial properties and have the ability to form complexes, which attract interest in providing new drug approaches. Thus, the incorporation in drug release systems is presented as a strategy to improve the antimicrobial activity of metallic compounds, as well as to enable a better solubilization and release of them. This work aimed to evaluate the activity of silver (I) coordination compounds: [Ag(PhTSC∙HCl)2](NO3)∙H2O (1), [Ag(HTSC∙HCl)2](NO3)∙H2O (2), [Ag(PhTSC∙HCl)2](Cl)∙H2O (3) and [Ag(HTSC∙HCl)2](Cl)∙H2O (4), unloaded and loaded into a polymeric nanoparticle (PN) against H. pylori ATCC 43504. The compound that showed the best activity (unloaded and loaded) was evaluated on the formation of mature H. pylori biofilm and biofilm and evaluated for cytotoxic potential on MRC- 5 and J774A.1 cell line, mutagenic potential and toxic potential in alternative in vivo model of Galleria mellonella. The PN composed of polymer (polycaprolactone), a surfactant (poloxamer 407) and an aqueous phase (phosphate buffer pH 7.4) was prepared by nanoprecipitation. The system characterization assays (dynamic light scattering, zeta potential and transmission electron microscopy (TEM)) showed that the PNs have particle sizes from 155.10 nm ± 3.60 to 180.0 nm ± 1.13, polydispersity index from 0.093 ± 0.028 to 0.266 ± 0.032 and zeta potential from -3.17 mV ± 0.359 to - 0.16 mV ± 0.045. The stability of the formulations was performed for 90 days, and in view of the MIC resultd, together with the stability, the nanoparticle incoporated with compound 1 (PN1) was selected as the best sample to follow with the other tests. TEM images confirmed the formation of PNs. Antibacterial activity was evaluated by the microdilution technique. The unloaded compound 1 had a Minimum Inhibitory Concentration (MIC) of 3.90 μg/mL and after loaded its action was potentiated to 0.78 μg/mL and a Minimum Bactericidal Concentration (MBC) of 7.81 μg/mL free for 1.56 μg/mL in PN1. For biofilm, PN1 showed better activity against compound 1, both in biofilm formation and in mature H. pylori biofilm. Biofilm formation was analyzed at different formation times (4, 24 and 36 hours) which was proposed as 4 h long enough for H. pylori to form the biofilm. In the cytotoxicity analysis, the system provided better results, providing H. pylori selectivity increase of about 7 times for MRC-5 and 3 times more when compared to J774A.1, in addition to PN1 not showing cytotoxicity against cell lines at the same concentration as MIC. PN1 allowed the inhibition of the mutagenicity presented by the free compound. In the in vivo assay on Galleria mellonella, the system allowed larvae survival at the maximum concentration prepared. This is the first demonstration of the activity of silver complexes in association with a polymeric nanoparticle against H. pylori and indicates a potential drug against H. pylori.
Descrição
Palavras-chave
Helicobacter pylori, Biofilme, Nanopartícula polimérica, Compostos de coordenação, Prata