S Our previous study first investigated feasibility of applying ultrasound (US) and microbubbles (MBs) via external auditory canal to facilitate drug delivery into inner ear. However, most drugs are in aqueous formulae and eliminated via Eustachian tubes after drug application. In this study, feasibility of sustained release of thermosensitive poloxamer 407 (P407)-based MB gel for US mediation-enhanced inner ear drug (dexamethasone, DEX) delivery was investigated. The sol-to-gel transition temperature showed that mixture of DEX and only 10% and 12.5% P407 in MBs can be used for in vitro and in vivo drug delivery experiments. In in vitro Franz diffusion experiments, the release rates of 12.5% P407-MBs + US groups in the model using DEX as the delivered reagent at 3 h resulted in values 1.52 times greater than those of 12.5% P407-MBs groups. In guinea pigs, by filling tympanic bulla with DEX in 12.5% P407-MBs (DEX-P407-MBs), USMB applied at post-treatment days 1 and 7 induced 109.13% and 66.67% increases in DEX delivery efficiencies, respectively, compared to the group without US. On the 28th day after US-mediated P407-MB treatment, the safety assessment showed no significant changes in the hearing thresholds and no damage to the integrity of cochlea or middle ear. These are the first results to demonstrate feasibility of US-modified liquid form DEX-P407-MB cavitation for enhancing permeability of round window membrane. Then, a gel form of DEX-P407-MBs was generated and thus prolonged the release of DEX in middle ear to maintain the therapeutic DEX level in inner ear for at least 7 days.

Ultrasound-induced microbubble (USMB) cavitation is widely used to promote drug delivery. Our previous study investigated USMB targeting the round window membrane by applying the ultrasound transducer to the tympanic bulla. In the present study, we further extended the use of this technology to enhance drug delivery to the inner ear by introducing the ultrasound transducer into the external auditory canal (EAC) or applying it to the skull. Using a 3-dimensional–printed diffusion apparatus mimicking the pathway for ultrasound passing through and reaching the middle ear cavity in vitro, the models simulating the transcanal and transcranial approach demonstrated 4.8-fold– and 3.7-fold–higher delivery efficiencies, respectively. In an in vivo model of guinea pigs, by filling tympanic bulla with microbubbles and biotin-FITC, USMB applied transcanally and transcranially induced 2.8-fold and 1.5-fold increases in biotin-FITC delivery efficiencies, respectively. In addition, the gentamicin uptake by cochlear and vestibular hair cells and gentamicin-induced hair cell loss were significantly enhanced following transcanal application of USMB. On the 28th day after transcanal USMB, safety assessment showed no significant changes in the hearing thresholds and the integrity of cochlea. These are the first results to our knowledge to demonstrate the feasibility and support the potential clinical application of applying USMB via EAC to facilitate drug delivery into the inner ear.

Noise-induced hearing loss (NIHL) is a common inner ear disease but has complex pathological mechanisms, one of which is increased oxidative stress in the cochlea. The high-mobility group box 1 (HMGB1) protein acts as an inflammatory mediator and shows different activities with redox modifications linked to the generation of reactive oxygen species (ROS). We aimed to investigate whether manipulation of cochlear HMGB1 during noise exposure could prevent noise-induced oxidative stress and hearing loss. Sixty CBA/CaJ mice were divided into two groups. An intraperitoneal injection of anti-HMGB1 antibodies was administered to the experimental group; the control group was injected with saline. Thirty minutes later, all mice were subjected to white noise exposure. Subsequent cochlear damage, including auditory threshold shifts, hair cell loss, expression of cochlear HMGB1, and free radical activity, was then evaluated. The levels of HMGB1 and 4-hydroxynonenal (4-HNE), as respective markers of reactive nitrogen species (RNS) and ROS formation, showed slight increases on post-exposure day 1 and achieved their highest levels on post-exposure day 4. After noise exposure, the antibody-treated mice showed markedly less ROS formation and lower expression of NADPH oxidase 4 (NOX4), nitrotyrosine, inducible nitric oxide synthase (iNOS), and intercellular adhesion molecule-1 (ICAM-1) than the saline-treated control mice. A significant amelioration was also observed in the threshold shifts of the auditory brainstem response and the loss of outer hair cells in the antibody-treated versus the saline-treated mice. Our results suggest that inhibition of HMGB1 by neutralization with anti-HMGB1 antibodies prior to noise exposure effectively attenuated oxidative stress and subsequent inflammation. This procedure could therefore have potential as a therapy for NIHL.

Gene therapy for sensorineural hearing loss has recently been used to insert genes encoding functional proteins to preserve, protect, or even regenerate hair cells in the inner ear. Our previous study demonstrated a microbubble- (MB-)facilitated ultrasound (US) technique for delivering therapeutic medication to the inner ear. The present study investigated whether MB-US techniques help to enhance the efficiency of gene transfection by means of cationic liposomes on HEI-OC1 auditory cells and whether MBs of different sizes affect such efficiency. Our results demonstrated that the size of MBs was proportional to the concentration of albumin or dextrose. At a constant US power density, using 0.66, 1.32, and 2.83 μm albumin-shelled MBs increased the transfection rate as compared to the control by 30.6%, 54.1%, and 84.7%, respectively; likewise, using 1.39, 2.12, and 3.47 μm albumin-dextrose-shelled MBs increased the transfection rates by 15.9%, 34.3%, and 82.7%, respectively. The results indicate that MB-US is an effective technique to facilitate gene transfer on auditory cellsin vitro. Such size-dependent MB oscillation behavior in the presence of US plays a role in enhancing gene transfer, and by manipulating the concentration of albumin or dextrose, MBs of different sizes can be produced.