Study On The Transfection Of Hydroxyapatite Nanoparticle Carried NT-3 Gene Via Intact Round Window Membrane Permeation

Objective:To explore the feasibility of hydroxyapatite nanoparticle carried NT-3 gene transfecting into auris interna through the intact round window membrane.To study whether the permeability of round window membrane to gene vector could be remarkably improved by the effect of facilitating agents.To observe the effect on cochlear structure and its function of hearing during the process of hydroxyapatite nanoparticle carried NT-3 gene transfecting into auris interna through the intact round window membrane by the effect of facilitation.Methods:Healthy white short-hair red-eye guinea pigs(n=31) were divided into following groups:hyaluronic acid pre-treatment group(Hya group,n=7) and histamine pre-treatment group(His group,n=6) as experimental groups;scala tympani drilling group(Drill group,n=3), liposome-DNA group(Lip group,n=6),direct tranfection group(PEI-HAT group,n=6) and blank group(n=3) as control groups.For experimental groups, after pre-treatment with hyaluronic acid or histamine on the intact round window membrane,a piece of gelfoam immersed with hydroxyapatite nanoparticles carrying NT-3 gene was placed on the round window membrane of the left ear for transfection.With preoperative and postoperative monitoring of ABR and DPOAE,the guinea pigs were killed after 72h.The perilymphs of left and right ears,the blood and cerebrospinal fluid of the animals were obtained for PCR amplification to detect the EGFP-DNA.The fluorescence of cochlear basilar membranes ttuough an improved film preparation method as well as the cochlear longitudinal slices by frozen sectioning was observed under fluorescence microscope.Results:Drilling method to the scala tympani has more damage on the hearing than methods through the intact round window membrane.By liposome mediation via the intact round window membrane,the permeability ratio of positive PCR testing is 3/6(postive PCR products / samples).Using 9.5mg/ml histamine as a facilitator,no positive PCR product was detected in the perilymph,but it was detected in the CSF.Use of 9.5mg/ml hyaluronic acid as facilation agent could make the permeability ratio of up to 6/7,and it was also detected in the contralateral ear and the ratio is 2/6.No postive detection was found in the blood.No significant green fluorescence was observed on the film-prepared basilar membranes.In the longitudinal slices, green fluorescence can be seen from the spiral ganglion and part of the basilar membrane,but it can not be determined whether the fluorescence is the result of gene transfection.Conclusion:(1) The transfection of therapeutical genes into auris interna through the intact round window membrane is feasibe,and it is a simple, effective and little-injury method.(2) Compared to the drilling method to the scala tympani,the permeation of drugs through the intact round window membrane is a safer method for clinical therapy.(3) Using hyaluronic acid as facilitating agent can effectively increase the permeability of the round window membrane,and thus raise the efficiency of transfection of foreign genes into internal ear.