Targeted Distribution In Vestibule And Pathway Entry Into Inner Ear Of Chitosan Nanoparticulate Drug Delivery System

Inner ear is one of the most complicated tiny organs in vertebrate animals,with the cochlear system responsible for hearing and the vestibular system contributing to balance control.Inner ear diseases are connected with pathological changes in cochlea,vestibule,and auditory nerve.Vestibular dysfunction is typically characterized by imbalance and vertigo,which seriously affects an individual's ability to perform activities of daily living or participate in work and leisure activities.Intratympanic delivery has been performed clinically to treat inner ear diseases.It has the advantages of avoiding the damage to delicate inner ear structures or functions and allowing a significantly lower risk of systemic adverse effects.However,delivery therapies to the inner ear have challenged otolaryngologists for a long time.Nanosized delivery vehicles are becoming of increasing interest and have been used for local application.It is well accepted that substance enters the perilymph from middle ear through the round window membrane(RWM),but the passage through the oval window(OW)has long been neglected.Up to now,researchers still know little about the pathway via which nanoparticles(NPs)enter the inner ear more effectively and the NP distributions in vestibular organs after local applications.Thus,we engineered Nile red-encapsulated chitosan(CS)NPs,investigated the NP distribution in three vestibular organs and further assessed the efficacy of NP transport via the RWM and OW pathways,which will lay a foundation for customizing a nanoparticulate delivery strategy for the effective treatment of peripheral vestibular diseases.Herein,we engineered fluorescence traceable CS NPs based on the ionic gelation.The DLS result of size distribution and TEM image showed NPs had a spherical shape and relatively uniform size,and there were homologous,intact particles in the perilymph after intratympanic injection of CS NPs.We first investigated the cellular distribution of Nile red-loaded CS NPs in the inner ear including three vestibular organs and two cochlear organs.Our results showed that there was prevailing level of CS NPs in the vestibular hair cells,dark cells and supporting cells,but negligible ones in cochlear hair cells and epithelial cells after intratympanic administration.Also,the stronger fluorescence signals of CS NPs were visualized and preferentially presented in the vestibular system,and the cochlear system exhibited a weaker signal after intratympanic injection of ten times concentrated CS NPs.Considering middle ear-RWM-scala tympani is a dominating pathway of substance entry into inner ear,we firstly speculated that hair cells and dark cells of the vestibule,compared to hair cells of the cochlea,might be more avid in accumulating NPs.Next,to explore the mechanism of the NP distributions in vestibule and cochlea,we utilized two cell models in vitro,normal cell line L929 and a conditionally immortalized cochlear auditory cell line derived from OC(HEI-OC1),and established inner ear explant cultures.Abundant CS NPs were localized in the two types of cells,and the uptake efficiencies in L929 and HEI-OC1 cells were further quantified by flow cytometry as 98.6% and 89.1%,respectively.Subsequently,we assessed whether NPs were able to enter the hair cells in cochlear explant cultures.Surface preparation of OC revealed stronger fluorescence signal around the hair cell nucleus,suggesting that CS NPs were capable of entering hair cells in the cochlear explants in vitro.We further speculated that the middle ear-OW-vestibule passage might dominate CS NPs entry into inner ear.To explore the availability of RWM and OW pathways to NP transport,we then constructed a nanoparticulate drug delivery system comprising poloxamer 407-based thermo-sensitive hydrogel loaded with CS NPs,allowing direct site-specific delivery of NPs to the RWN or stapes footplate.NIRF imaging,HPLC quantitative study and CLSM imaging clearly revealed that CS NPs entered the inner ear through both the RWM and OW,but the latter,as a royal gate,afforded a convenient access to facilitate CS NPs transport into vestibular organs.To investigate the biocompatibility of CS NPs,we assessed the cytotoxicity of NPs against the two cell models.The viability of L929 and HEI-OC1 cells was 84.9%~97.1% and 92.9%~97.7%,suggesting that CS NPs were biocompatible with normal cells.In addition,we assessed the morphological changes of inner ear by HE stain and CLSM imaging,revealing that CS NPs may be generally safe to vestibule and cochlea tissues.Overall,our study firstly suggested that the OW,as a more effective pathway than the RWM route,dominantly controlled delivery of CS NPs to vestibule,casting a new light on future clinical applications of NPs in the effective treatment of vestibular disorders by minimizing the risk of hearing loss,ultimately opening an avenue for treating peripheral vestibular diseases more specifically and efficiently.