Exosome Packaging of the Targeted Antioxidant CAT‐SKL to Protect the Sensory Cells in the Inner Ear

Hearing loss is a chronic health condition that affects millions of people worldwide. In addition to age‐related hearing loss, excessive exposure to loud noise or ototoxic chemotherapy drugs (e.g., cisplatin) can also lead to hearing impairment via oxidative stress in the sensory cells in the inner ear (cochlea). In such cases, the endogenous antioxidant enzymes (e.g., catalase) in the cochlea that normally mitigate the accumulation of reactive oxygen species become overwhelmed, resulting in permanent cell damage. To combat this oxidative stress, our research program is working to find effective strategies to deliver therapeutic doses of exogenous catalase to the cochlea. In the present study, we aimed (1) to evaluate whether our recombinant derivative of catalase, CAT‐SKL (which is designed to target the peroxisomes of cells) could protect against cisplatin‐induced cell death in vitro , and (2) to package CAT‐SKL into exosomes, and determine the safety, distribution and efficacy of this targeted antioxidant in vivo . In our first experiments, we assessed the safety and protective effects of CAT‐SKL using cochlear‐derived cells (HEI‐OC1) and WST‐1 cell viability assays. As predicted based on our previous studies in other tissues (e.g., brain, retina), CAT‐SKL was not harmful to HEI‐OC1 cells, and actually improved cell viability at higher concentrations. Importantly, CAT‐SKL offered significant protection against cisplatin‐induced ototoxicity, even when the doses of cisplatin exceeded levels known to cause damage. Motivated by this in vitro otoprotection, we then endeavored to package CAT‐SKL into exosomes, as this would provide a strategy to deliver the novel pharmaceutical in vivo . We packaged CAT‐SKL into macrophage‐derived exosomes (exoCAT‐SKL) via sonication, and purified the loaded exosomes using size exclusion chromatography. Successful loading of exoCAT‐SKL was confirmed using Western blotting and H202 decomposition assays. Next, adult mice were delivered intranasal administrations of a biotinylated version of CAT‐SKL packaged in exosomes, and their brains were then harvested and processed for streptavidin immunohistological staining. We observed extensive labelling of exoCAT‐SKL in the brains of these mice; evidence that intranasal administration not only effectively introduced our therapeutic into systemic circulation, but it was able to surpass tissue specific barriers that usually hinder uptake of therapeutics. Our ongoing experiments are investigating the bioavailability of repetitive intranasal dosing of exoCAT‐SKL in adult mice by harvesting their organs, and performing histopathological assessments. Once we have established the bioavailability of exoCAT‐SKL in the cochlea following intranasal delivery, we will be poised to investigate its efficacy at attenuating cisplatin‐induced ototoxicity in vivo . Based on our findings, we suggest that CAT‐SKL is a promising therapeutic for protection of cochlear cells from cisplatin‐induced ototoxicity, and that packaging CAT‐SKL into exosomes may offer an effective way to deliver therapeutic doses of this targeted antioxidant to cells undergoing oxidative stress. Support or Funding Information BrainsCAN, CIHR, The W. Garfield Weston Foundation This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

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