Modification Of Hydroxyapatite Nanoparticle-Based Gene Vector And The Experimental Study On Its Gene Transfection In Inner Ear

Objective:1) To investigate the biological properties of hydroxyapatite nanoparticles surface-modified by polyethylenimine (PEI-nHAT), and the DNA-binding and DNA protection capacity of PEI-nHAT in vitro.2) To investigate the safety and effectiveness on transfection of PEI-nHAT carrying enhanced green fluorescent protein with neurotrophin-3(pEGFPC2-NT3) in the cochlea via intact round window membrane (RWM).3) To investigate the safety and effectiveness on transfection of PEI-nHAT-pEGFPC2-NT3in the vestibular system via intact RWM.Methods:1) nHAT were synthesized by a chemical coprecipitation-hydrothermal synthesis method. Transmission electron microscopy (TEM) observation and Zeta potential detection were carried out after surface modification with PEI. Dispersibility and stability of PEI-nHAT were also observed, the capacity of PEI-nHAT on DNA-binding and DNA protection against nuclease digestion were assessed in different concentration as well.2) Eight normal adult chinchillas of both sexes were used in this study. The right ears from six animals were used as the experimental group for local application of PEI-nHAT-pEGFPC2-NT3onto the intact RWM, and the left were treated with the same volume of empty vector (PEI-nHAT) as self control. Both ears from another two animals were treated with the same volume of normal saline as blank control. The tone burst-evoked Auditory Brainstem Responses (ABR) and Distortion Product Otoacoustic Emissions (DPOAE) were recorded in1kHz,4kHz,8kHz,12kHz and16kHz pre-operation and48h post-transfection. Chinchillas were sacrificed after auditory function evaluation. The temporal bones containing cochleae were fixed for surface preparations and cryosections. Immunohistochemistry and immunofluorescence staining were carried out to observe the morphologic changes and localize the EGFP expression in cochlea under confocal microscope.3) Eight normal adult chinchillas of both sexes were used in this study. The right ears from six animals were used as the experimental group for local application of PEI-nHAT-pEGFPC2-NT3onto the intact RWM, and the left were treated with the same volume of empty vector (PEI-nHAT) as self control. Both ears from another two animals were treated with the same volume of normal saline as blank control. The vestibular samples were fixed and then prepared as surface preparations and cryosections after animals were sacrificed. The morphologic observations of vestibular organs and localized observation of EGFP expression were carried out after immunohistochemistry and immunofluorescence staining.Results:1) After surface modification with PEI, the shape of PEI-nHAT was similar to a short rod with uniform size. The PEI-nHAT showed good dispersivity and stability in solution. The mean size (Mean±SD) of PEI-nHAT was73.09±27.32(nm). At pH7, the Zeta potential of PEI-nHAT was42.46±10.9mV at the concentration of250μg/ml. PEI-nHAT showed good capacity of DNA-binding and DNA protection against DNase I digestion at concentrations from125μg/ml to2000μg/ml.2) Neither significant threshold shift in tone burst-evoked ABR nor significant amplitude changes in DPOAE occurred at all tested frequencies between pre-operation and48h post-transfection in each group (P>0.05). The cells in cochlear basilar membrane, stria vasularis and cochlear spiral ganglions appeared morphologically normal after48h transfection in each group. EGFP expression was detected in some outer pillar cells in the organ of Corti, a few marginal cells in stria vascularis, some cells in Rosenthal's canal in the PEI-nHAT-pEGFPC2-NT3transfected group, although the transfecton efficiency in cochlea was not high.3) There was no vestibular dysfunction occurred such as step instability, head tilting, rolling or circling after48h transfection in each group. Vestibular hair cells and the supporting cells appeared morphologically normal after48h transfection in each group. Abundant, condensed green fluorescence was found in the zone of dark cells and transitional zone on both sides of the crista and around the macula of the utricle. Scattered EGFP signals were also detected in vestibular hair cells.Conclusion:1) PEI-nHAT are positive charged and have good dispersivity and stability;2) PEI-nHAT have good capacity of DNA-binding and DNA protection against nuclease digestion at concentrations from125μg/ml to2000μg/ml.3) The approach of gene transfer through intact RWM has no affect on the morphology of cochlea or auditory function, which can serve as an safe and effective way of transferring gene into cochlea and vestibule.4) Vestibular dark cell is the specific targeted cell of PEI-nHAT for gene therapy in the inner ear. It might have the ability of actively uptaking PEI-nHAT-pEGFPC2-NT3complex, although the detailed mechanism is not clear.5) PEI-nHAT are innogarnic materials which could be a potential vector of gene therapy for the inner ear diseases, especially for the vestibular disorders.