Publication
Development of a new approach to control otitis media pathogens
| Summary: | Otitis media is one of the most common illnesses in childhood and the leading cause for an antibiotic prescription. Endolysins, peptidoglycan hydrolases encoded by bacteriophages, are an attractive antibiotic alternative. Their transtympanic delivery directly to the middle ear would maximize bioavailability, reducing the probability of a recurrent or chronic infection. However, the tympanic membrane acts as a barrier, preventing the diffusion of molecules from the ear canal to the middle ear. Delivery systems with permeation enhancing characteristics would allow surpassing this barrier and, at the same time, provide sustained and targeted delivery. Therefore, the main goal of this work was to develop a novel delivery system for a non-invasive transtympanic treatment of otitis media using endolysins. For this, four objectives were addressed: i) identification of the bacterial pathogens causing otitis media in middle ear fluid samples collected in Braga, Portugal; ii) characterization of endolysins active against otitis media pathogens; iii) encapsulation of endolysins for a controlled delivery; iv) evaluation of the transtympanic permeation ability of the developed formulation. Pathogens in middle ear fluid samples collected from children with otitis media were identified by culture and quantitative polymerase chain reaction. Haemophilus influenzae was the most prevalent pathogen, followed by Streptococcus pneumoniae. Endolysins against both pathogens were identified, but only the endolysin from the S. pneumoniae phage MS1 (MSlys) was successfully expressed. MSlys exhibits high specificity towards S. pneumoniae, killing different otitis media isolates and showing promising antimicrobial properties against planktonic and biofilm cells. The MSlys endolysin was encapsulated into deformable liposomes composed of L-alpha-lecithin and sodium cholate (L:SC:MSlys) or PEG2000 PE (L:PEG:MSlys) with an average efficiency of about 35%, being released in a sustained manner over time. Furthermore, the MSys-loaded liposomes showed no cytotoxicity against fibroblast and keratinocyte cell lines and interacted with S. pneumoniae cells. Ex vivo transtympanic permeation studies showed that PEGylated liposomes significantly increased the transport of MSlys through the tympanic membrane compared to its free form and maintained antipneumococcal activity after the initial permeation time points. Nevertheless, degradation of MSlys occurred after an extended incubation period at 37 ºC, which consequently affected its antimicrobial effectiveness. In summary, endolysin-loaded PEGylated liposomes are a promising approach for transtympanic treatment of otitis media. Nonetheless, further optimization is required to expand the overall therapeutic efficacy of this novel strategy to control otitis media pathogens. |
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| Main Authors: | Silva, Maria Daniela Ferreira |
| Subject: | Endolysin Liposomes Otitis media Transtympanic Endolisina Lipossomas Otite média Transtimpânico |
| Year: | 2022 |
| Country: | Portugal |
| Document type: | doctoral thesis |
| Access type: | open access |
| Associated institution: | Universidade do Minho |
| Language: | English |
| Origin: | RepositóriUM - Universidade do Minho |
| Summary: | Otitis media is one of the most common illnesses in childhood and the leading cause for an antibiotic prescription. Endolysins, peptidoglycan hydrolases encoded by bacteriophages, are an attractive antibiotic alternative. Their transtympanic delivery directly to the middle ear would maximize bioavailability, reducing the probability of a recurrent or chronic infection. However, the tympanic membrane acts as a barrier, preventing the diffusion of molecules from the ear canal to the middle ear. Delivery systems with permeation enhancing characteristics would allow surpassing this barrier and, at the same time, provide sustained and targeted delivery. Therefore, the main goal of this work was to develop a novel delivery system for a non-invasive transtympanic treatment of otitis media using endolysins. For this, four objectives were addressed: i) identification of the bacterial pathogens causing otitis media in middle ear fluid samples collected in Braga, Portugal; ii) characterization of endolysins active against otitis media pathogens; iii) encapsulation of endolysins for a controlled delivery; iv) evaluation of the transtympanic permeation ability of the developed formulation. Pathogens in middle ear fluid samples collected from children with otitis media were identified by culture and quantitative polymerase chain reaction. Haemophilus influenzae was the most prevalent pathogen, followed by Streptococcus pneumoniae. Endolysins against both pathogens were identified, but only the endolysin from the S. pneumoniae phage MS1 (MSlys) was successfully expressed. MSlys exhibits high specificity towards S. pneumoniae, killing different otitis media isolates and showing promising antimicrobial properties against planktonic and biofilm cells. The MSlys endolysin was encapsulated into deformable liposomes composed of L-alpha-lecithin and sodium cholate (L:SC:MSlys) or PEG2000 PE (L:PEG:MSlys) with an average efficiency of about 35%, being released in a sustained manner over time. Furthermore, the MSys-loaded liposomes showed no cytotoxicity against fibroblast and keratinocyte cell lines and interacted with S. pneumoniae cells. Ex vivo transtympanic permeation studies showed that PEGylated liposomes significantly increased the transport of MSlys through the tympanic membrane compared to its free form and maintained antipneumococcal activity after the initial permeation time points. Nevertheless, degradation of MSlys occurred after an extended incubation period at 37 ºC, which consequently affected its antimicrobial effectiveness. In summary, endolysin-loaded PEGylated liposomes are a promising approach for transtympanic treatment of otitis media. Nonetheless, further optimization is required to expand the overall therapeutic efficacy of this novel strategy to control otitis media pathogens. |
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