Peptides rapidly transport antibiotic across the intact tympanic membrane to treat a middle ear infection

Abstract

The tympanic membrane (TM) forms an impenetrable barrier to medical therapies for middle ear (ME) diseases like otitis media. By screening a phage-displayed peptide library, we have previously discovered rare peptides that mediate the active transport of cargo across the intact membrane of animals and humans. Since the M13 filamentous bacteriophage on which the peptides are expressed are large (nearly 1 µm in length), this offers the possibility of noninvasively delivering drugs, large drug packages, or gene therapy to the ME. To evaluate this possibility, EDC chemistry was employed to covalently attach amoxicillin, or neomycin molecules to phage bearing a trans-TM peptide, as a model for large drug packages. Eight hours after application of antibiotic-phage to the TM of infected rats, ME bacterial titers were substantially reduced compared to untreated animals. As a control, antibiotic was linked to wild-type phage, not bearing any peptide, and application to the TM did not affect ME bacteria. The results support the ability of rare peptides to actively deliver pharmacologically relevant amounts of drugs through the intact TM and into the ME. Moreover, since bacteriophage engineered to express peptides are viral vectors, the trans-TM peptides could also transport other viral vectors into the ME.

Keywords: Otitis media; active transport; antibiotics; middle ear; otic delivery; transtympanic drug delivery.

Figure 1.

(A) Schematic reconstructed representation of M13mp phage vector engineered to express a peptide (in green), whose gene is ligated into the pIII gene (in red). Each bacteriophage particle has five pIII coat protein molecules. In contrast, the cylindrical body of the phage, shown substantially shortened here for display purposes, contains thousands of pVIII coat protein molecules on its surface (in yellow). The helical pIX protein (in black) is at the other cap of the phage. The engineered peptide is displayed at the free N-terminus of pIII protein filaments. (B) Schematic diagram: Linkage of antibiotic to phage using EDC-mediated coupling (image from Yacoby et al., 2007). The red stars represent the antibiotic molecules conjugated with pVIII phage protein via EDC chemistry. For illustration, the carboxyl side chains at the N-terminal helix of pVIII major coat protein are bound to three molecules of neomycin.

Figure 2.

Phage recovery from the middle ear for nine trans-TM peptide phage, reflecting a range of peptide transport rates (adapted from Kurabi et al., 2016). The initial titers were standardized at 10⁹ PFU/μL. This study utilized the TMT3 synthetic peptide and peptide phage.

Figure 3.

Linkage to TMT3 synthetic peptide does not affect antibiotic activity. Amoxicillin (AMX) and ciprofloxacin (CIP) antibiotics were linked to the TMT3 peptide, and the activity against NTHi in liquid culture was evaluated. Recovery of NTHi after 8 h in liquid culture media with antibiotic alone or antibiotic linked to TMT3 peptide. The MIC is defined as the lowest concentration of test drug at which the % inoculum growth was maintained inhibited.

Figure 4.

Growth curves of NTHi upon treatment with different TMT3 phage-antibiotic conjugated constructs in culture. Neomycin (Neo) and amoxicillin (AMX) molecules crosslinked to bacteriophage retained anti-bacterial activity, but ciprofloxacin (CIP) showed reduced activity. The absorbance level of the bacteria (growth) was measured by monitoring O.D. A₆₀₀.

Figure 5.

Antibiotics linked to a trans-TM peptide 1:1 and applied to the TM of an infected ear had no effect on NTHi titers. The initial inoculum concentration of NTHi applied into the middle ear of rats is ∼5x10³ CFUs and the drug constructs AMX linked to TMT3 peptide or CIP linked to TMT3 peptide were applied for 4h in vivo on the intact tympanic membrane. The NTHi recovery in the ME were determined from plate counts of formed NTHi colonies in the middle ear fluid (CFUs).

Figure 6.

Control of middle earbacterial infection by phage-mediated antibiotic delivery across the tympanic membrane (TM). Application of neomycin (Neo) or amoxicillin (AMX) conjugated to a trans-TM phages TMT3 on the intact TM reduced bacterial titers in an infected middle ear compared to without treatment (NTHi alone), or with exposure of the TM to WT phage conjugated to antibiotics. The ciprofloxacin-TMT3 phage (TMT3 + CIP) did not have an effect on NTHi titer since its antibiotic activity was compromised after the EDC crosslinking reaction (see Figure 4).