Cellular Uptake And Transport Mechanism Of Nano-delivery Systems With Different Characteristics Via Inner Ear Administration:In Vitro And In Vivo Evaluation

Sensorineural hearing loss and tinnitus, vertigo are considered as three major refractory disease in otology, which seriously influence human health with an increasing incidence rate. For the treatment of inner ear disorders, inner ear administration is a better choice in comparison to the traditional systemic administration, but the particle size,surface chemical properties etc. of drug delivery systems can affect the drug distribution in the inner ear,and the drug delivery systems may also cause cochlear disease. Here, we compared the performance of nano-delivery systems with different characteristics in optimizing the delivery to inner ear both in vitro and in vivo. It is desirable to provide a reference for the application of nano-delivery systems for the treatment of inner ear diseases.In order to investigate the uptake and distribution of NPs with various sizes,poly(lactic/glycolic acid) nanoparticles(PLGA NPs) was used as a model. In our study, we found that all NPs with different size including <100 nm, 100-200 nm and 200-400 nm of PLGA NPs had no significant cytotoxicity when incubated with L929 cells, and all of them could be taken up by the cells and distribute around the nucleus. Quantitative analysis suggested that the NPs with medium size showed the greatest cellular uptake, followed by small size and large size, while the uptake were 99.5%, 97.6% and 71.9% respectively. In vivo histocompatibility and distribution experiments indicated that PLGA NPs with different sizes showed good biocompatibility, and all of them can be delivered to cochlea ofthe inner ear and distribute to the various tissues, while the NPs with medium size possessed the largest distribution.PLGA NPs with medium size were modified with poloxamer 407(P407), chitosan(CS)and methoxy poly(ethylene glycol)(mPEG) respectively. Here, we undertook a systematic comparison of PLGA NPs with or without surface modification of hydrophilic molecules for optimizing the delivery to inner ear especially the outer hair cells(OHCs) both in vitro and in vivo. We found that all examined PLGA NPs are preferably biocompatible with L929 cells and HEI-OC1 cells, and can be uptaken by them, while P407-PLGA NPs showed the highest cellular uptake( L929: 96.1%; HEI-OC1: 91.4%). In vivo experiments also proved all examined PLGA NPs have good biocompatibility and can be transported to the inner ear.And the surface-modified NPs reached the organ of Corti at a higher level, while P407-PLGA NPs showed the prominent fluorescence in cochlear imaging. In cochlea tissue distribution experiments, all examined PLGA NPs can be transported to the cochlea and PLGA NPs with different modified can reach the OHCs, especially P407-PLGA NPs. The results of fluorescence semi-quantitative analysis suggested that the mean fluorescence intensity of P407-PLGA NPs taken up by OHCs was 1.95, 1.76, 1.39-fold compared to that of the unmodified, CS- and mPEG-PLGA NPs, respectively(All P<0.001).CS NPs was used as a model to study the effects of different metal complexes of CS NPs on toxicity and uptake both in vitro and in vivo, which was unmodified CS NPs, copper complexes of CS NPs(Cu-CS NPs), calcium complexes of CS NPs(Ca-CS NPs), zinc complexes of CS NPs(Zn-CS NPs), iron complex of CS NPs(Fe-CS NPs) respectively.The cytotoxicity experiments exhibited good biocompatibility at low concentrations(<50μg/mL), then the cytotoxicity increased as the concentration increased. In vitro L929 cellular uptake experiments, unmodified and different metal complexes of CS NPs except Fe-CS NPs had a great uptake(>90%), while in HEI-OC1 cellular uptake experiments,unmodified and different metal complexes in the CS NPs can be taken up by the cells, and the uptake of unmodified CS NPs and Zn-CS NPs were higher than others. In vivo animal experiments, all CS NPs except Ca-CS NPs exhibited good biocompatibility, and all ofthem can be distributed to the organizations of cochlea.In summary, the study showed that nanoparticles with different characteristics would influence the biocompatibility, uptake and distribution in vivo and in vitro. Adjusting the characteristics of nano systems may lead a better drug delivery system. When PLGA nanoparticles system is applied to the inner ear administration, adjusting the size among100-200 nm may be more suitable for the cellular uptake. Nanomedicine strategy using modified PLGA NPs, especially that employing P407 decoration, will significantly improve their uptake and distribution. We also provided reference for the optimization of the prescription design for the application of CS nano system to the inner ear in that the different metal coordination will affect its biocompatibility and distribution behavior.