Abstract
We previously identified peptides that are actively transported across the intact tympanic membrane (TM) of rats with infected middle ears. To assess the possibility that this transport would also occur across the human TM, we first developed and validated an assay to evaluate transport in vitro using fragments of the TM. Using this assay, we demonstrated the ability of phage bearing a TM-transiting peptide to cross freshly dissected TM fragments from infected rats or from uninfected rats, guinea pigs and rabbits. We then evaluated transport across fragments of the human TM that were discarded during otologic surgery. Human trans-TM transport was similar to that seen in the animal species. Finally, we found that free peptide, unconnected to phage, was transported across the TM at a rate comparable to that seen for peptide-bearing phage. These studies provide evidence supporting the concept of peptide-mediated drug delivery across the intact TM and into the middle ears of patients.
Highlights
Persistent OM produces conductive hearing loss during critical periods of language acquisition[11] and may result in fibrosis and scarring that permanently damage the conductive apparatus of the middle ear (ME): 15–20% of teenagers exhibit visible tympanic membrane (TM) scarring due to prior OM12
When trypan-blue was added to the upper assay device chamber, the dye was excluded from the lower chamber by an intact TM fragment
A major motivation for the development of our chamber assay was to determine whether TM fragments could be used in vitro to test trans-TM transport of peptides, since in vivo testing of human TMs is not possible
Summary
Persistent OM produces conductive hearing loss during critical periods of language acquisition[11] and may result in fibrosis and scarring that permanently damage the conductive apparatus of the middle ear (ME): 15–20% of teenagers exhibit visible tympanic membrane (TM) scarring due to prior OM12. Using the technique of phage display, we recently discovered unique peptides that can transport large (1 μm in length and 7 nm in diameter) phage particles across the intact TM of rats undergoing bacterial OM31 This active transport could form the basis for local drug delivery to the ME without the need for surgery or injections through the TM, both of which require general anesthesia in children. We developed an in vitro chamber assay system, in which fragments of the TM from various species can be tested before proceeding to human translational work This was used to determine the rate of peptide phage transport observed for the rat TM with that across the TMs isolated from guinea pigs, rabbits and humans postmortem
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