Desenvolvimento e caracterização de lipossomas de ramnolipídeos encapsulados com peptídeos inibidores de topoisomerases bacterianas
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Data
2018-03-15
Autores
Sanches, Beatriz Cristina Pecoraro
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O sistema ParE-ParD é um sistema toxina-antitoxina (TA) onde ParE atua como a toxina e ParD a antitoxina. ParE tem comprovada atividade de inibição do sítio catalítico das enzimas DNA girase e Topoisomerase IV (Topo IV), sendo esta uma atividade que pode ser aproveitada para fins terapêuticos. ParELC3, um peptídeo derivado à ParE também apresenta esta habilidade de inibição enzimática in vitro, contudo, experimentos também comprovam a dificuldade de permeabilidade e alta complexidade estrutural que este material apresenta, o que pode ser um problema para sua aplicação para fins terapêuticos. Assim, com a intenção de viabilizar o potencial biotecnológico deste peptídeo, foi feita sua incorporação em lipossomas compostos majoritariamente por ramnolipídeos. Este biotensoativo, produzido pela rota fermentativa da Pseudomonas aeruginosa, foi selecionado por ser considerado “sustentável”, ter atividade antimicrobiana intrínseca e ainda ser pouco empregado para fins nanotecnológicos. Ensaios de caracterização físico-química e morfológica, via espalhamento de luz dinâmica (DLS), microscopia eletrônica e transmissão (MET) e microscopia eletrônica de transmissão-crio (MET- Crio) dos lipossomas foram realizados e comprovou-se a formação de bicamadas unilamelares. Ensaios de eficiência de encapsulação mostraram que os sistemas lipossomais incorporaram entre 44 e 61% de ParELC3. Em relação à atividade antimicrobiana, as versões de ParELC3 incorporado nos lipossomas de ramnolipídeos foram mais eficientes frente a E. coli e S. aureus quando comparados à atividade antimicrobiana de ParELC3 livre. Realizou-se estudos de estabilidade e de liberação prolongada do sistema lipossomal, e comprovou-se que os lipossomas prolongam a liberação de ParELC3, mantendo a estabilidade média dos mesmos por até 20 dias. Mostrou-se que na concentração dos experimentos, os lipossomas compostos de ramnolipídeos não são tóxicos para células HepG2, assim como o peptídeo ParELC3 livre. Acredita-se que com o lipossoma obtido com ParELC3 pode ser utilizado no combate às infecções bacterianas.
ParE-ParD system is a toxin-antitoxin (TA) system where ParE acts as a toxin and ParD as an antitoxin. ParE can inhibit the catalytic sitium of DNA gyrase and Topisomerase IV (Topo IV) enzymes, which can be useful for pharmaceutics applications. ParELC3, an analogue peptide from ParE also has this in vitro enzymatic inhibition ability, but, has also a permeability difficult and high structural complexity, which can be a problem for its therapeutical application. So, pretending to use the biotechnological potential of this peptide, its internalization in liposomes composed mainly by rhamnolipids was proposed. This biosurfactant, produced by the fermentative route of Pseudomonas aeruginosa, was selected for being considered "sustainable", for its intrinsic antimicrobial activity and low toxicity, in addition to not being employed for nanotechnological experiments. Physic-chemistry and morphological characterization assays were done by dynamic light scattering (DLS), transmission electronic microscopy (TEM) and transmission electronic microscopy – Cryo (TEM – Cryo), then, bilayers production was comproved. Encapsulation efficiency tests showed that liposomal systems can incorporate efficiently between 44 e 61% of ParELC3. In relation to the antimicrobial activity, versions of internalized ParELC3 into liposomes composed mainly by rhamnolipids were more efficient than compared to antimicrobial activity for free ParELC3, both for E coli and S aureus bacterias representers. In function of these promising results, was proposed to do stability and drug release assays, and results showed that liposomes prolonged the ParELC3 liberation and them average stability is around 20 days. The results still showed that at the concentration tested, liposomes constituted mainly by rhamnolipids are not toxic to HepG2 cells, neither for ParELC3 peptide. We believe that these systems, where liposomes are encapsulated with ParELC3, can be used against bacterial infections.
ParE-ParD system is a toxin-antitoxin (TA) system where ParE acts as a toxin and ParD as an antitoxin. ParE can inhibit the catalytic sitium of DNA gyrase and Topisomerase IV (Topo IV) enzymes, which can be useful for pharmaceutics applications. ParELC3, an analogue peptide from ParE also has this in vitro enzymatic inhibition ability, but, has also a permeability difficult and high structural complexity, which can be a problem for its therapeutical application. So, pretending to use the biotechnological potential of this peptide, its internalization in liposomes composed mainly by rhamnolipids was proposed. This biosurfactant, produced by the fermentative route of Pseudomonas aeruginosa, was selected for being considered "sustainable", for its intrinsic antimicrobial activity and low toxicity, in addition to not being employed for nanotechnological experiments. Physic-chemistry and morphological characterization assays were done by dynamic light scattering (DLS), transmission electronic microscopy (TEM) and transmission electronic microscopy – Cryo (TEM – Cryo), then, bilayers production was comproved. Encapsulation efficiency tests showed that liposomal systems can incorporate efficiently between 44 e 61% of ParELC3. In relation to the antimicrobial activity, versions of internalized ParELC3 into liposomes composed mainly by rhamnolipids were more efficient than compared to antimicrobial activity for free ParELC3, both for E coli and S aureus bacterias representers. In function of these promising results, was proposed to do stability and drug release assays, and results showed that liposomes prolonged the ParELC3 liberation and them average stability is around 20 days. The results still showed that at the concentration tested, liposomes constituted mainly by rhamnolipids are not toxic to HepG2 cells, neither for ParELC3 peptide. We believe that these systems, where liposomes are encapsulated with ParELC3, can be used against bacterial infections.
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Palavras-chave
Peptídeos, Sistema toxina-antitoxina, Ramnolipídeos, Peptides, Toxin-antitoxin systems, Rhamnolipids, Lipossomas, Liposomes, Antimicrobian activity, Atividade antimicrobiana