| Part â… The safety and efficacy of the CGP-hydrogel-gentamicin for Meniere's diseaseObjective:Evaluate the safety and efficacy of the CGP-hydrogel-gentamicin delivery system for Meniere's disease. Hope to develop a novel drug delivery system which could control vertigo successfully and lower hearing loss.Methods:C57BL/6J mice,6-8month-old, were divided into three groups (A, B, and C). The mice in group A were injected intratympanically with the gentamicin sulfate solution (6mg/ml, lOul) every other day. In group B, the mice were injected intratympanically with the same quantity and concentration of gentamicin every2days. In the groupC, gentamicine laden CGP-hydrogel(300mg/ml,0.2ul) was placed in the round window niche of mice. Then the rotating drum (16rpm) test was used to assess the vestibular function every day after the first injection, and auditory brainstem response (ABR) threshold was measured to evaluate auditory function before the first injection and at14th day after first injection.Results:Auditory Function Assessment show:On postoperative day14, ABR threshold of mice in group A is significantly improved at16kHz,24kHz,32kHz,40kHz than that in pre-injection (p<0.05); ABR threshold of mice in group B is not significantly improved at16kHz,24kHz,32kHz than that in pre-injection (p>0.05), except at40kHz (p=0.030, p<0.05); ABR threshold of mice in group C is not significantly improved at16kHz,24kHz,32kHz,40kHz than that in pre-injection (p>0.05).Vestibular Function Assessment:After first injection, the mean remaining times of the mice in group A demonstrated statistical decreases on postoperative day10(p=0.031, p<0.05), however, there was no significant differences compared to the presurgery values on postoperative day14(p=0.105, p>0.05). In group B, there were no significant differences on postoperative day6,10,14(p>0.05) in contrast with the pre-surgery data. The mice in group C demonstrated a gradual decline of the mean holding times on postoperative day6, day10, day14(p=0.041,0.034,0.006, respectively; p<0.05). The mean remaining times of the mice in group C demonstrated statistical decreases compared to group A (p=0.019; p<0.05) and group B (p=0.016; p<0.05) on postoperative day14.Conclusions:The mice in group A showed balance dysfunction on postoperative day10, and followed by a balance function recovery on postoperative day14, meanwhile, they were caused a significant hearing loss at all frequency; the mice in group B didn't cause significant balance dysfunction, but were caused hearing loss at40kHz; in contrast, the mice in group C yielded noticeable and stable balance dysfunction from day6to day14without significant hearing loss at all frequency. So CGP-hydrogel carried with gentamicin could establish a stable and enduring destruction of the vestibular function without causing significant hearing loss. The CGP-hydrogel is a controlled and sustained local drug delivery system to the inner ear. Part IIThe distribution of gentamicin in inner ear after local administration via a Chitosan glycerophosphate hydrogel delivery systemObjectives:Recently, we engineered a novel chitosan glycerophosphate (CGP)-hydrogel based local drug delivery system, which can offer sustained drug release (e.g., gentamicin or dexamethasone) to the inner ear. However, the precise target sites, in which the drugs interacted with cells in the cochlea and vestibule where they produced their intended effects using this system, still remain unclear. The goal of this study was to examine the distribution of gentamicin in the inner ear after the delivery by CGP-hydrogel system and the change of the morphology of hair cells, so as to determine how gentamicin affected the function of inner ear.Methods:A matrix of CGP-hydrogel loaded with gentamicin conjugated to Texas Red (GTTR), which was used as a tracer, was injected into round window niche (RWN) of the left ear of C57/BL6mice. Keeping the left ear of the mice up for half an hour. The dosage of the gel which was injected into RWN is0.2μl CGP-GTTR-hydrogel containing40μg gentamicin base. The mice were killed and fixed day1and day7post-injection. Confocal fluorescence microscopy was used to determine the localization of gentamicin in cochlear and vestibular system. All the images were processed by MetaMorph Software (Universal Imaging Corporation, West Chester, PA). The software reported the intensity as a grayscale value.Results:The intensities of GTTR fluorescence in grayscale units in basal turn and in medial turn at day1were51.59±6.42,20.64±4.14, respectively. The intensity of GTTR in basal turn was significantly stronger than that in medial turn (p<0.01) at day1. Similarly, the intensities of the fluorescence in basal turn and medial turn at day7were43.01±3.61and12.17±2.43, respectively (p<0.01). Negligible fluorescence was observed in apical turn both at day1and day7. Meanwhile, comparing the intensity of GTTR in the same turn between the different time points, less intensity was detected at day7than that at day1both in basal turn (p<0.05) and in medial turn (p<0.05). Moreover, there was some outer hair cells loss in the basal turn at day7, no hair cells loss in medial and apical turn at any time points. Simultaneously, intensities of GTTR in hair cells of macula of saccule at day1and day7were14.13±4.09,4.69±1.76, respectively. The intensity of GTTR at day1was significantly stronger than that at day7(p<0.05). In addition, the number of hair bundles on top of the cuticular plate at day1was obviously more than that at day7.Conclusions:The distribution of gentamicin distributes in inner ear via CGP-hydrogel delivery system in a time-and basal to apical-dependent manner. Thus, this system possesses two characteristics, i.e., the longer the hydrogel was injected into RWN, the less intensity of the GTTR fluorescence can be detected; the farther distance from the RWN, the less intensity of the GTTR fluorescence can be detected. These two characteristics account for high frequency hearing loss and vestibular dysfunction using this system to deliver gentamicin into inner ear.
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