The Inner Ear Delivery Of MiR96 Based On The CMC-TAT Nanocarriers

BackgroundSensorineural hearing loss is result from the injury of cochlear hair cells, auditory nerve and nerve centre, which induce the disorder of sensing sounds and the transmission of nerve impulses. Most of sensorineural hearing loss resulte from the abnormal structure or function of epithelial hair cells, which cannot regenerate in mammals. The fundamental thrapy is to facilitate the restore of abnormal hair cells or regeneration of hair cells. With the development of gene technology, researchers have found about 30424 mature miRNA which involve proliferation, differentiation, growth in almost all cells, and a variety of which highly expressed in mice. The miRNA 183 family were closely related to the cochlear hair cell differentiation, growth and human hearing loss.RNA interference is mainly about silencing specific gene. The key point of RNA interference is to choose optimal carrier of miRNA for the inner ear.Common vectors include viral and non-viral vectors, the former which risk the immune responses and potential carcinogenic effects, limite the application in vivo.In recent years, non-viral vectors have become focus. Nanoparticles as non-viral miRNA carriers are appropriate vectors because of their biocompatibility and non-immunogenicity.Liposome is the most common non-viral carrier of miRNA due to its high transfection rate, but the cytotoxity and the lack of targeted and controlled release because of its postive charge prevent it from applying in clinic.CMC-TAT nanocarriers (CTNs) are pH-responsive nanoparticles generated by self-assembly approach.CMC(Carboxymethyl chitosan) is biocompatible, nontoxitic, organism absorbable and water-soluble. The TAT peptide is derived from transcription activating factor TAT of HIV (human immunodeficiency virus), which can effectively interact with nucleic acids via covalent bonds or non-covalent electrostatic interactions. TAT peptide can efficiently mediates the transport of exogenous biological macromolecules across the biological membrane and barrier. CTNs can efficiently transfect cells and reverse their negative charge to positive charge upon exposure to acid stimulation, which provide a series of desired functions to nanoparticles such as strengthened endosomal escape ability and achieve the controlled release. The liposome is the most common non viral vector for inner ear delivery of miRNA, although CTNs has been successfully used in the miRNA transfection of tumor cells, and can achieve the regulatory function, CTNs for the inner ear delivery of miRNA are not reported.ObjectiveTo construct nanometer carrier for inner ear delivery of miRNA,and to explore the efficiency and security of CTNs for miR96 by transfect 293 cells and cochlear b asilar membrane, which lay the foundation for the functional study of miR96 in the inner ear.Method1. The Lipo-miR96-cy3 complexes are generated by mixing liposomes with miR96 and transfect 293 cells and cochlear basilar membrane at different concentrition (30nM, 50nM, 100nM), and blank and miR96-cy3 control groups are set up separately.Observe the fluorescence intensity of cy3 and cell-specific distribution by the laser scanning confocal microscope and detect the cytotoxity of liposomes by MTT test.2. The CTNs are generated with the optimal weight ratio of 1.5:1 (CMC:TAT), which are used for the incorporation with miR-96(M-CTNs).3. Transfect 293 cells and cochlear basilar membrane with Lipo-miR96-cy3 complexes and M-CTNs at the concentrition of 150nM, and blank control group is set up separately. To compare the efficiency, security and cytotoxity of the vectors mentioned above.Result1. Lipo-miR96-cy3 complexes are uptaken by 293 cells after incubation for 8 hours.The cells with red fluorescence increased along with the increase of concentrition of Lipo-miR96-cy3 complexes. The red fluorescence postive cells of 50nM are significant higher than that of 30nM (t=-10.801, P=0.000), the red fluorescence postive cells of 100nM are significant higher than that of 50nM (t=11.754, P=0.000). There is no red fluorescence in the control group. Red fluorescence are observed in the inner hair cells, outer hair cells, supporting cells and spiral ganglion cells after incubation with cochlear basilar membrane for 8 hours. The red fluorescence intensity increased along with the increase of concentrition of Lipo-miR96-cy3 complexes with non-specific cell distribution.2. M-CTNs with a size of 186.8 nm and a zeta potential of 30.9 mV are successfully prepared in this part. The encapsulation efficiency of M-CTNs can achieve above 90% and reverse their negative charge to positive charge at the pH 5.5.3. The red fluorescence of M-CTNs is weaker than that of Lipo-miR96-cy3 complexes in both 293 cells and cochlear basilar membrane.ConclusionCationic liposome mediated miR96 transfection efficiently in vitro, but its cytotoxity prevent it from application in clinic.The transfection efficiency of M-CTNs is lower than that of liposomes, and CTNs still need to be further optimized.