# Transcranial ultrasound combined with intravenous metformin-loaded oxygenated microbubbles attenuates noise-induced hearing loss in mice

## TL;DR

A new treatment combining ultrasound and metformin-loaded bubbles helps protect mice from hearing loss caused by loud noise.

## Contribution

A dual-action strategy using oxygenated microbubbles and metformin with ultrasound for targeted cochlear protection against noise-induced hearing loss.

## Key findings

- US-mediated cMet-OMBs increased intracochlear oxygen tension after noise exposure.
- Metformin reduced ROS production via mitochondrial complex I inhibition and antioxidant pathway activation.
- Mice treated with cMet-OMBs + US showed better hearing preservation and hair cell survival.

## Abstract

Noise-induced hearing loss (NIHL) involves a biphasic pathophysiology. Intense noise exposure causes immediate cochlear vasoconstriction and ischemia, leading to transient hypoxia. Subsequent reperfusion triggers excess reactive oxygen species (ROS) production, resulting in oxidative stress and hair cell injury. This study therefore developed two oxygenated albumin microbubble (OMB) formulations—ionic-bond metformin-coated (iMet-OMBs) and covalent-bond metformin-encapsulated (cMet-OMBs)—and combined them with transcranial ultrasound (US) to enhance targeted delivery to the cochlea. This approach aims to provide transient oxygen supplementation while simultaneously reducing ROS-mediated injury. Microbubbles were characterized for morphology, oxygen loading, and metformin content. Based on their superior stability and drug-loading profile, cMet-OMBs were selected for in vivo evaluation. In a mouse NIHL model, animals were administered cMet-OMBs systemically via retro-orbital injection, followed by US triggering over the temporal bone. Auditory brainstem response (ABR) thresholds, cochlear oxygen tension, and outer hair cell (OHC) survival were assessed. US-mediated cMet-OMBs rupture transiently increased intracochlear oxygen tension, counteracting early hypoxia after noise exposure. Metformin released from cMet-OMBs attenuated ROS production through mitochondrial complex I inhibition and antioxidant pathway activation. Mice treated with cMet-OMBs + US showed significantly lower ABR threshold shifts and better OHC preservation compared with controls. This dual-action strategy combines transient oxygen supplementation from OMBs with sustained antioxidant protection from metformin. While oxygen delivery raises intracochlear oxygen tension, metformin suppresses ROS generation through mitochondrial complex I inhibition and AMPK/Nrf2 activation. This controlled, US-triggered release achieves net cochlear protection against NIHL without excessive oxidative burden.

Metformin-coated (ionic bond) and metformin-encapsulated (covalent bond) oxygenated albumin microbubbles (iMet-OMBs and cMet-OMBs) were both prepared and characterized. In mice, cMet-OMBs were selected and injected into the blood. Transcranial ultrasound-mediated cavitation fragments cMet-OMBs within the cochlear microvasculature, transiently increasing BLB permeability and locally releasing oxygen. This targeted oxygen delivery, combined with metformin’s antioxidant properties, elevates intracochlear oxygen partial pressure while suppressing ROS, thereby protecting hair cells from noise-induced damage.