On-Demand Synthesis of Antiseptics at the Site of Infection for Treatment of Otitis Media

Abstract

Otitis media (OM) is the main reason for pediatric antibiotic prescriptions. The current treatment mandates a rigorous regimen of multidose antibiotics over 5-10 days. The systemic antibiotic exposure and often prematurely terminated treatment due to the challenge of drug administration to young patients are believed to breed antibiotic resistance. To address these challenges, we designed a local treatment that converted a metabolic product (H₂O₂) of an OM pathogen (Streptococcus pneumoniae) into a potent antiseptic (HOBr), a reaction catalyzed by locally administered vanadium pentoxide nanowires. The therapeutic, HOBr, was only synthesized in the presence of the pathogen, enabling on-demand generation of therapeutics for OM treatment. Hypohalous acids are broad-spectrum and have a long history in general disinfection applications without breeding substantial drug resistance. A single dose of the nanowire formulation eradicated OM in a standard chinchilla model in 7 days with no observable tissue toxicity or negative impact on hearing sensitivity.

Keywords: Vanadium pentoxide nanowire; antiseptics; haloperoxidase-like activity; on-demand synthesis; otitis media.

Fig. 1.. Characterization of the V₂O₅ NWs.

(A, B) TEM and HRTEM images of the V₂O₅ NWs. (C) FFT of the HRTEM image of a single V₂O₅ NW. (D-F) the STEM image (D) and the corresponding EDX elemental mapping of V (E) and O (F). (G) XPS survey scan of the V₂O₅ NWs. (H) High-resolution XPS spectra of the V₂O₅ NWs with deconvoluted core level spectra of O(1s) (blue peaks) and V(2p) (orange peaks). The black solid line corresponds to the envelope/fitting curve, and the black dots represent the raw data.

Fig. 2.. Catalytic activities of the V₂O₅ NWs.

(A) Scheme of the oxidative bromination reaction (converting PR to Br₄PR), used to quantify the reaction kinetics of HOBr generation. (B) UV-vis spectra demonstrating the bromination reaction only in the presence of V₂O₅ NWs, Br⁻, and H₂O₂. The reaction mixtures contained 25 μM PR, and/or 0.04 mg/mL V₂O₅ NWs, and/or 1 mM Br⁻, and/or 100 μM H₂O₂; the spectra were collected in triplicates after 12 hours of reaction time. (C) Dependence of the initial rate of the bromination reaction on the concentration of V₂O₅ NWs, collected at the Br⁻ concentration of 1 mM and the H₂O₂ concentration of 10 μM. (D) Dependence of the initial rate of the bromination reaction on the concentration of Br⁻, collected at the V₂O₅ NWs concentration of 0.04 mg/mL and the H₂O₂ concentration of 10 μM. (E) Dependence of the initial rate of the bromination reaction on the concentration of H₂O₂, collected at the V₂O₅ NWs concentration of 0.04 mg/mL and the Br⁻ concentration of 1 mM. The initial concentration of PR was kept at 25 μM for C, D, and E. (F) Dependence of the initial rate of the bromination reaction on pH, where the concentration of V₂O₅ NWs was 0.04 mg/mL, of Br⁻ was 1 mM, and of H₂O₂ was 10 μM. (G) Stability of the V₂O₅ NWs over 12 hours, where the concentration of V₂O₅ NWs was 0.04 mg/mL, of Br⁻ was 1 mM, and of H₂O₂ was 10 μM. Data are mean ± SD. n = 4.

Fig. 3.. Antibacterial effect of the V₂O₅ NWs.

(A) Bacterial growth curves of S. pneumoniae. The OD600 was monitored for 15 hours, where the initial concentration of V₂O₅ NWs was 0.04 mg/mL, of Br⁻ was 1 mM, and of H₂O₂ was 20 μM. Data are mean ± SD. (n = 4). ns, not statistically significant, **p<0.01, ****p<0,0001; all comparisons were made with respect to the “Nontreated” group. (B) Images of the blood agar plates obtained by plating the S. pneumoniae cultures at the end of the incubation shown in (A).

Fig. 4.. Biocompatibility assessments in vitro and in vivo.

(A) Confocal microscopy images of the LIVE/DEAD staining of hFBs and PC12. Green, live cells; red, dead cells. Cells were incubated with 0.01 mg/mL or 0.04 mg/mL V₂O₅ NWs (as labeled in the figure), 1 mM Br⁻, and 10 μM H₂O₂ for 24 hours. (B) Cell survival rate (%), calculated by counting the live and dead cells in (A). Data are mean ± SD. n = 4. ns, not statistically significant, ****p<0.0001. (C) ABR thresholds in response to an acoustic click before and after the treatment with a formulation containing 0.08 mg/mL V₂O₅ NWs and 1 mM Br⁻. The black dashed line indicated the hearing threshold of healthy chinchillas before the treatment. Data are mean ± SD. (n = 5). (D) The cross-section micrographs of the H&E-stained pristine and treated TMs. The treated TMs were exposed to 0.08 mg/mL V₂O₅ NWs and 1 mM Br⁻ for 7 days.

Fig. 5.. In vivo therapeutic efficacy of the V₂O₅ NWs in a standard chinchilla OM model.

(A) The timeline of the in vivo procedures to induce and treat OM in chinchillas. (B) Otoscope images of the TMs of healthy (left) and infected (right) animals. The red arrows indicated the bulging TM and erythema observed during an OM episode. (C) Time course of the bacterial CFU count in the MEF of animals with OM. The formulations contained 0.08 mg/mL V₂O₅ NWs and/or 1 mM Br⁻. Data are mean ± SD. n=8 for the nontreated group; n=5 for the group that received both V₂O₅ NWs and Br⁻; n=4 for the group that only received V₂O₅ NWs. Log₁₀CFU for CFU ~0 was set to zero instead of minus infinity for the purpose of illustration. **p<0.01. (D) Vanadium content in the MEF of animals with OM, following a single intrabullae injection quantified using ICP-MS (normalized to Sulfur (S), a surrogate for total tissue quantity). Data are mean ± SD. n = 3-4 for each time point. (E) Representative H&E-stained sections of pristine, infected, and treated TMs. (F) Thicknesses of the TMs, quantified using the H&E-stained sections. Data are mean ± SD. n = 5. ns, not statistically significant, **p<0.01, ****p<0.0001

Fig. 6.

Schematic diagram of antimicrobial properties of the V₂O₅ NWs for the treatment of otitis media.