목적: This study aims to develop an optogenetic microneedle system for minimally invasive neuro-stimulation of the inner ear. The microneedle is designed to deliver AAV viral vectors efficiently to the cochlea via the round window membrane, enabling targeted expression of optogenetic proteins in auditory neurons. By comparing microneedle- mediated delivery with conventional microcatheter injection, we seek to determine the efficacy and safety of this novel approach. 방법:A biodegradable hyaluronic acid-based microneedle was fabricated to release AAV viral vectors directly into the cochlea’s scala tympani. In vitro experiments were conducted using assembloids, a combination of cochlear progenitor organoids and spiral ganglion neurons, which were incubated with microneedles containing AAV-mCherry-Halorhodopsin. Epifluorescence analysis was performed to confirm viral transfection, and multielectrode array (MEA) recordings were used to assess neuronal responses to light stimulation. For in vivo validation, rats were divided into experimental groups receiving either AAV-GFP via microneedle or conventional viral injection. Western blot analysis was conducted to quantify AAV-GFP expression in cochlear tissue. Additionally, optogenetically engineered rats underwent auditory brainstem response (ABR) testing using a continuous 532 nm laser (0.38 mW) to evaluate functional stimulation. 결과:Western blot analysis revealed that microneedle-mediated AAV-GFP delivery resulted in significantly higher GFP expression compared to conventional injection, indicating improved efficiency of viral transfection. Epifluorescence analysis showed strong transfection of spiral ganglion neurons in the microneedle group, whereas conventional delivery primarily transfected inner hair cells. In assembloid models, AAV-mCherry-Halorhodopsin was successfully expressed in both inner hair cells and neurons. When subjected to light stimulation, these assembloids exhibited robust phase-locked electrical activity, confirming functional optogenetic responses. In vivo experiments demonstrated that rats receiving microneedle- mediated AAV-mCherry-Halorhodopsin delivery exhibited laser-induced ABR responses without acoustic stimuli. These responses were characterized by irregular but increased amplitude waveforms upon laser irradiation, suggesting successful neural activation through optogenetic stimulation. 결론:This study demonstrates that optogenetic microneedles can effectively deliver viral vectors to cochlear target cells with high efficiency and specificity. The ability of microneedle-delivered optogenetic proteins to induce neural responses upon light stimulation suggests a viable alternative to conventional electrical stimulation methods. These findings support the further development of bioengineered microneedle systems for non-invasive auditory neuro-stimulation, potentially reducing the need for surgically implanted mechanical devices.