Preventive Effect And Mechanism Study Of A666 Peptide-conjugated Stealth Nanoparticles By Cisplatin-induced Hearing Loss

Objective: This study aimed to investigate the feasibility of sustained dexamethasone(DEX)delivery to the inner ear following round window membrane(RWM)administration mediated by A666-polyethylene glycol-coated polylactic acid(A666-DEX-NP)nanoparticles and explore the efficacy of dexamethasone loaded A666-DEX-NP nanoparticles against cisplatin-induced hearing loss by RWM administration.Methods: DEX was fabricated into PEG-PLA nanoparticles(DEX-NP)using emulsion and evaporation technique,and then A666-(CLEPRWGFGWWLH)peptides was connected to this DEX-NP through covalent bond between maleinimid and sulfydryl.A666 can specifically bind to prestin,which is limited to the outer hair cells(OHCs).The pharmaceutical properties of A666-DEX-NP including connection efficiency of A666,theparticle size,Zeta potential,size morphology,encapsulation efficiency(EE),drug loading(DL),were examined through XPS,DLS,TEM and HPLC,respectively.House Ear Institute-Organ of Corti and cisplatin-treated guinea pigs(12 mg/kg,i.p.)were used as in vitro and in vivo models for investigating the targeting and protective efficiency against cisplatin by CLSM.The molecule mechanism of the protect against cisplatin-induced ototoxicity was observed via immumohistochemical staining and western blot.Results: The results showed that ideal A666-DEX-loaded nanoparticles can be obtained by conjugated A666-(CLEPRWGFGWWLH)peptide to DEX loaded nanoparticles(DEX-NPs),which were fabricated by emulsion and evaporation technique.A666-DEX-NPs had a hydrodynamic diameter of 157.80 ± 14.33 nm,and zeta potential of-32.53 ± 0.64 m V,The DL and EE were 1.20 ± 0.33% and 0.44 ±0.12%,respectively.A666-coumarin 6-NP could be significantly internalized by HEI-OC1 cells and especially target and accumulate in outer hair cells through the tightly combined between A666 peptide and prestin.In addition,the A666-DEX-NP locally applied onto RWM of guinea pigs have a continuous release of DEX in 48 h,significantly longer than the free DEX that was cleared out within 8 h after RWM administration via the same dose.Furthermore,pretreated with A666-DEX-NP 1h for guinea pigs before cisplatin-treated can effectively preserved OHCs and showed significant hearing protection at 4 k Hz,8 k Hz,and 16 k Hz as compared to pretreatment with saline,DEX,or DEX-NP formulation.More important,we infer that A666-DEX-NPs protect cisplatin-induced hearing loss via decreasing reactive oxygen species levels,inhibiting the activation of caspase-3 and enhancing the express of Blc-2。Conclusion: In this study,we synthesized A666 peptide decorated,DEX loaded,OHC targeting stealth nanoparticles(A666-DEX-NP)for the first time.A series of in vitro and in vivo targeting and sustained release properties were studied.This study confirmed that A666-DEX-NP has good external hair cell targeting properties and is an ideal carrier for continuous delivery of drugs in the inner ear.At the same time,A666-DEX-NP can effectively antagonize hearing loss induced by cisplatin at 4 k Hz,8 k Hz and 16 k Hz frequencies.This study will provide new ideas and strategies for the prevention and treatment of cisplatin induced ototoxicity.