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.