Introduction
Otitis media (OM), the infection and subsequent inflammation of the middle ear, is the most common illness within the first 24 months of birth1. By age 5, over 95% of children in the U.S. have had at least one episode of OM2,3. In particular, 58% OM episodes are due to bacterial infections caused by gram-positiveStreptococcus pneumoniae (S. pneumoniae ) and/or gram-negative non-typeable Haemophilus influenzae(NTHi)4,5, pathogens that commonly colonize the nasopharynx and invade the auditory bullae opportunistically to cause OM6.
Oral antibiotic therapy is the current mainstay of treatment for OM. A typical course of treatment comprises 7-10 days of multidose antibiotic regimens7. As a result, OM represents the most common reason for pediatric antibiotic prescriptions written to US children2,3. The level of systemic antibiotic exposure caused by OM is further exacerbated by identified antibiotic resistance of OM pathogens. For example, S. pneumoniae , responsible for over 30% of all OM cases, is known to have greater tolerance for ß-lactam and macrolide8,9. Even with effective fluoroquinolones such as ciprofloxacin, the minimum inhibitory concentration (MIC) of S. pneumoniae is as high as 0.5 – 4 µg/mL10,11. Effective eradication of S.pneumoniae OM requires high antibiotic concentrations in the middle ear, sustained throughout the treatment by adhering to the rigorous multidose oral regimens. The high levels of systemic antibiotic exposure often cause side effects, such as diarrhea, vomiting, and oral thrush12, which in turn make it challenging to continue the treatment and potentially lead to recurrent OM and wide-spread antibiotic resistance.
In this report, silver nanoparticles (AgNPs) were examined as a potential treatment for the OM pathogens. Contrary to small-molecule antibiotics, we found AgNPs [stabilized with polyvinylpyrrolidone (PVP)] to be high efficacious against S. pneumoniae , with MICs lower than that of NTHi, showing a great potential as a broad-spectrum therapy for OM. In recent years, AgNPs have become an attractive alternative to antibiotics due to their excellent antibacterial effects against both gram-positive and gram-negative pathogens13 and even bacteria with multidrug resistance14. Several mechanisms have been considered to explain the antimicrobial efficacy of AgNPs15. In brief, AgNPs have been observed to attach to the cell membrane of bacteria, leading to critical damages such as membrane penetration and disabled membrane functions such as respiration (due to deactivation of membrane-bound essential enzymes such as respiratory chain dehydrogenases)16,17, which in turn increases bacterial membrane permeability18,19. AgNPs that penetrated a bacterial cell can damage DNA and deactivate intracellular enzymes18,20, leading to rapid cell death21. Furthermore, AgNPs are known to generate reactive oxygen species (ROS) including superoxide anion (O2•−), hydroxyl radical (OH), and hydrogen peroxide (H2O2)22. The excess ROS produced by AgNPs often deplete glutathione (GSH), an antioxidant produced by virtually all living organisms23,24, and subsequently damage cell membrane and intracellular organisms23,24. Nevertheless, AgNPs have been demonstrated to cause minimal cytotoxicity or immunological responses25 and have thus been adopted across a range of biomedical applications, including drug delivery (e.g., wound healing26, eye infection caused by Pseudomonas aeruginosa 27, and post-cardiac surgery mediastinitis28) and medical imaging (e.g., human oral cancer29 and multimodality cancer30). Although efficacy of AgNPs against OM pathogens has not been studied previously, we hypothesized that AgNPs could be highly potent, especially against the resistant bacteriaS. pneumoniae . That hypothesis was based on the potential synergistic interactions between AgNPs and H2O2 due to their Fenton-like reactions and the H2O2-generating ability ofS. pneumoniae 31–33.
A hydrogel delivery system was designed to enable the localized and sustained presence of AgNPs during the course of the treatment. This design enables an AgNPs-containing formulation to be administered through a perforated tympanic membrane as a liquid, which quickly turns into a firm solid gel to achieve sustained antimicrobial effects. Reverse thermal gelation, the property that enables liquid-phase administration of the formulation at room temperature and rapid gelation at elevated temperature (e.g., body temperature), was achieved using poloxamer 407 (P407)34,35. It enables a single-dose administration into the middle ear with ease and, once in place, prolonged presence of the formulation to prevent recurrent OM. Furthermore, P407 has been tested as mucoadhesive formulations in rectal delivery of a range of therapeutics such as tizanidine HCl (TIZ) (for treatment of spasticity)36, Ibuprofen (for treatment of pain, fever, rheumatoid arthritis and osteoarthritis)37, and quinine in children38 (for treatment of malaria), in the nasal delivery of selegiline hydrochloride (for treatment of Parkinson’s disease)39 and opiorphin40 (for treatment of acute and chronic pain), and vaginal delivery of itraconazole41 and clotrimazole42(for treatment of vaginal candidiasis). No observable irritation to the mucosal membrane has been observed43, hinting at the compatibility of P407-based formulations with the middle ear mucosa. Furthermore, delivery of OM treatments through a perforated tympanic membrane is particularly applicable to OM patients with recurrent episodes. A recent study showed 54.85% chronic OM cases are accompanied by tympanic membrane perforations44, whereas among children with recurrent AOM 92% had tympanic membrane perforations45. Therefore, the AgNPs reported here have the potential to enable a single-dose and sustained treatment for OM.
In this report, we obtained stable AgNPs colloidal solutions by reducing Ag+ in the presence of stabilizer polyvinylpyrrolidone. The as-synthesized particles were ~10 nm in diameter, as demonstrated using DLS and TEM. Upon successful demonstration of their antimicrobial efficacy in vitro using S. pneumoniae , NTHi, and Streptococcus mutans(S. mutans ) and biocompatibility using human fibroblast and PC12 Adh cell line (a pheochromocytoma cell line used to test neurotoxicity), the particles were further incorporated in an 18% (w/v) P407 aqueous solution, yielding a hydrogel with reverse thermal gelation temperature at around 25ºC. The hydrogel maintained high antimicrobial efficacy and biocompatibility. Therefore, the formulation reported here has the potential to eradicate bacterial pathogens of OM without antibiotics, which circumvents the systemic antibiotic exposure and associated harmful side effects caused by the current oral antibiotic therapy in OM treatment.