Protective Effect Of PLGA Nanoparticles Targeting Prestin Of Outer Hair Cells Against Acute Hearing Loss

As the most common sensory disorder worldwide,hearing loss(HL)is the clinical manifestation of many inner ear diseases.The damage or loss of inner ear sensory hair cells(HCs)is the main cause of HL,but the inner ear of mammalian lacking the ability of proliferation or regeneration.Therefore,damage or loss of HCs in the inner ear can lead to permanent hearing loss,which is difficult to recover.At present,the application of nano-drug delivery system in the inner ear has been widely studied,showing the great potential of delivering various therapeutic agents to the inner ear.However,how to effectively deliver drugs to HCs of apical cochlear turns is a major challenge for otologists.It was generally believed that after local administration,drugs would gather in HCs of basal cochlear turns,and then slowly spreaded to the HCs of middle and apical cochlear turns in the perilymph of tympanic scala,which mainly played the role of prevention and treatment of high-frequency HL.However,it is not clear how to design a drug delivery system to effectively target HCs of apical cochlear turns to prevent low-frequency HL.Prestin is a transmembrane protein specifically expressed on the lateral membrane of outer hair cells(OHCs)of inner ear,which enhances the motor property of OHCs and improves hearing sensitivity.Therefore,active targeting of the cochlear OHCs is an effective strategy.In this study,PLGA NPs were used as delivery carriers and modified with targeting peptide or hydrophilic substance(P407).Firstly,the targeting properties of PLGA NPs in vivo were studied,and then the effects of histone deacetylase inhibitors(HDACi)-encapsulated PLGA NPs on acute hearing loss in guinea pigs were investigated,expected to provide a new idea for the treatment of clinical hearing loss.Hererin,we prepared coumarin-6-loaded PLGA NPs by the classical emulsification-solvent volatilization method.The uptake characteristics of PLGA NPs were investigated in the HEI-OC1 cell model in vitro.The results showed that all NPs could be effectively taken up by cells,but the A665 modified PLGA NPs were significantly distributed in the nucleus,indicating that the A665 could promote the uptake of NPs.Then,the distribution of NPs in the round window membrane(RWM)and basement membrane of cochlea was investigated by two local administration methods in vivo.PLGA NPs was the most distributed in the RWM after RWN administration,while the other three groups had little and no significant difference.The distribution of A665 modified NPs was significantly different from unmodified NPs on in the apical turns of basement membrane(BM).Meanwhile,the distribution in the basal turns and apical turns of A665-PLGA NPs and A665/P407-PLGA NPs was significantly different.In addition,the same trend was observed after direct injection through the RWM.Previous studies have generally shown that NPs were aggregated and distributed in basal and middle turns of HCs in BM,so we speculated that A665 has the ability to target HCs of the apical turns in BM.In order to further study the distribution of the drug delivery system in the BM,Rhodamine B modified with A665 and coumarin-6 loaded NPs were used as tracers,and the distribution of A665 modified NPs in OHCs of BM was further studied by high-resolution laser confocal scanning microscope.The results showed that regardless of the drug administration methods,the distribution trend of A665 modified NPs in the BM was basal turns < middle turns < apical turns.There was a reverse distribution trend,which indicated that prestin protein might be more distributed in OHCs of the apical turns of BM.Subsequently,in order to study the targeting mechanism of NPs,we used paraffin sections and immunofluorescence to investigate the mechanism in the whole level and cell level of cochlear tissue.It was worth noting that the NPs modified with A665 would accumulate in the organ of Corti.Immunofluorescence studies of A665 and prestin protein revealed that the targeting mechanism was mediated by prestin protein.Next,to explore the potential application of targeted nano-drug delivery system in the treatment of HL,two histone deacetylation inhibitors(curcumin and sodium butyrate)were encapsulated.Then the oto-protective effects of the drug-loaded NPs were investigated through guinea pig model of acute hearing loss.Drug-loaded NPs showed significant oto-protective effects at all detection frequencies(4 k Hz,8 k Hz,click).Espartically,A665 modified curcumin-loaded NPs showed significant differences(P < 0.05)compared with CUR API or unmodified NPs.Combined with the morphological analysis of inner ear hair cells,the NPs provided almost complete oto-protective effects.However,the effects of sodium butyrate loaded-NPs was lower than sodium butyrate sodium butyrate API,but the A665 modified NPs could significantly reduce the hearing threshold and reduce the loss of inner ear hair cells,suggesting the potential clinical application value of A665 modified NPs in the treatment of HL.In order to investigate the safety of targeted nano-drug delivery system in the treatment of HL,the cytotoxicity of blank nanocarrier was investigated by using HEI-OC1 cells,which showed good cytocompatibility.Then,zebrafish embryos were used as toxicity study model to evaluate the effects of all NPs on the hatching rate,survival rate,teratogenic rate,body length and cumulus hair cells of zebrafish embryos within 96 h,.The results showed that there was no obvious toxicity of zebrafish embryos.In vivo guinea pig model,the safety of the targeted nano-drug delivery system for inner ear drug delivery was investigated through auditory function and morphological evaluation.The results showed that the targeted nanometer drug delivery system had good biosafety,suggesting that it could be used as a potential therapeutic agent for clinical application.In summary,this study demonstrated that A665 modified PLGA NPs could target prestin protein of OHCs in cochlea,and HDACi-loaded targeting PLGA NPs could provide almost complete oto-protective effects for HL induced by kanamycin combined with furosemide.This study is the first time to study the effect of HDACi-loaded PLGA NPs on acute hearing loss in guinea pigs,and to provide reference for clinical treatment of hearing loss in the future.