The Transport Mechanism Of Nanoparticulate Drug Delivery System Across The Round Window Membrane

Topical administration of medicines via the round window membrane(RWM),the primary interfaces to the inner ear,is favored by otologists due to its efficacy and noninvasive delivery,the advantage of bypassing the blood–perilymph barrier,simultaneously the reduced toxicity to the nontargeted tissues compared to systemic administration.Nowadays,extensive literatures have reported the application of nanoparticles for inner ear diseases therapy which has become one of the primary strategies to conquer the biological barrier due to the small particle size,high specific surface area and the high activity.Previous studies on the application of nanomedicine in the inner ear were mostly emphasized on inner ear tissue distribution of nanoparticles(NPs),the pharmacokinetics and pharmacodynamics study,but seldom on the interaction between the NPs and the RWM,the primary interfaces to the inner ear.Unraveling the specific mechanism of NPs across the RWM would be a key point for the rational design of inner ear nano-drug delivery system with better therapeutic effect.Over the last couple of decades,the application of the Poly(lactic-co-glycolic acid)(PLGA)in the drug delivery system was reported overwhelmingly as it is the FDA approved biodegradable drug delivery vector.The NPs containing coumarin-6 were prepared by classical emulsifying solvent evaporation method.Coumarin-6 was incorporated as a tag for the PLGA NPs to make them traceable in tissues.We used confocal laser scanning microscope(CLSM)to visualize the distribution of PLGA NPs in the RWM and the TEM images of perilymph further demonstrate that PLGA NPs could transport through the RWM into the perilymph.The time dependence investigation of PLGA NPs across the RWM showed that the coumarin-6 fluorescence intensity in the RWM increased gradually as time progressed over the period of 10 min to 30 min,then faded at 60 min.Raising the concentration of PLGA NPs would increase the coumarin-6 fluorescence amount in the RWM which revealed that the entry of PLGA NPs into the RWM in a concentration dependent manner and the most NPs were found in RWM at 0.09 g/ml.As seen from the TEM images of the RWM after intratympanic injection of PLGA NPs for 30 min,a series of endocytic pits,endocytic vesicles,exocytic release and the sealed tight junction illustrated the vesicular transport pathway of the NPs across the RWM,restricting the paracellular pathway.The endocytic mechanism of the PLGA NPs across the cell membrane of the RWM was classically analyzed by means of various endocytic inhibitors,which revealed that the PLGA NPs were internalized predominantly via a macropinocytosis and caveolin-mediated endocytic mechanism.To further achieve the passage loci of the NPs in the RWM,the colocalization analysis and Golgi-related inhibitors proved that the NPs were entrapped within the lysosomal compartments and/or the endoplasmic reticulum/Golgi apparatus which mediated the exocytic release of the NPs.The concentration of drug in the inner ear depends greatly on the time of drug coming into contact with the RWM.Chitosan(CS),a biodegradable polymer,is a good drug carrier with bioadhesive properties.Simultaneously,chitosan or its derivatives based NPs can open the tight junctions transiently and reversibly.The chitosan nanoparticles encapsulating coumarin-6 were prepared by ionic crosslinking method.A 3D reconstruction images of the RWM and TEM images of the perilymph revealed directly that the NPs could passage through the RWM into perilymph.The time and concentration dependent experiment showed that the fluorescence intensity of the coumarin-6 in the RWM was increased gradually as time progressed over the period of 0 min to 60 min,then faded from 60 min to 240 min,while the transport of CS NPs across the RWM was in concentration dependent way.Substance may cross the epithelium barriers along two ways: transcellular or paracellular pathway.As shown from the TEM images of the RWM after intratympanic injection of CS NPs for 60 min,a series of active uptake events which involved both endocytic and exocytic processes and the opened tight junction in RWM revealed that the transcellular and paracellular pathway both involved in the CS NP entry into the RWM.The transport mechanisms of the CS NPs across the RWM were investigated with different endocytosis inhibitors.Our data corroborated that the macropinocytosis,caveolin-mediated endocytosis,clathrin-mediated endocytosis were responsible for the endocytosis of CS NPs in the RWM,which means the nonspecific interaction between CS NPs and the RWM.In this study,the colocalization analysis of CS NPs with lysosome made us envision that endocytosed NPs could be entrapped in the lysosomal compartment.The Golgi related inhibitors revealed that the ER/Golgi and Golgi/cell membrane pathways could mediate the exocytosis of CS NPs.Taken together,we first demonstrated directly that the PLGA NPs and CS NPs could transport through the RWM into perilymph and clarified the specific mechanism among the endocytosis,intracellular trafficking and exocytosis process.Meanwhile,this study was the first attempt to depict a clear picture of the delivery of PLGA NPs and CS NPs in the RWM which will provide a guideline for optimizing nano-vehicle delivery across the RWM into the inner ear.