A666多肽介导主动靶向隐形纳米粒对顺铂耳毒性预防作用及机制研究

In sensorineural hearing loss the main characteristic pathological change is the permanent and irreversible hair cell loss. With the lack of effective therapeutic strategies after hearing loss occurred, the prevention becomes crucial. According to the published study, the PEG-PLA stealth nanoparticle can permeate through round window membrane (RWM) and have significant protective effect against cisplatin-induced ototoxicity in low-frequency region (4, 8 kHz). The protective effects in high-frequency region (16, 24 kHz), however, is not observed. The limited aggregation of non-targeted nanoparticle in the organ of Corti suggest that active-targeted nanoparticle may be the resolution. Here, we develop a novel nanoparticulate DDS, A666 peptide conjugated, prestin targeted nanoparticles for dexamethasone (A666-DEX-NP), as a model to investigate the its protective effect against cisplatin-induced ototoxicity and its functional mechanism. Our preliminary results demonstrate that A666-DEX-NP selectively bind to outer hair cells both in vitro and in vivo. To our knowledge, the approach of using A666 peptide to bind prestin is the first time such approach is applied with cochlea targeted nanoparticulate DDS. Our research may provide a new strategy for the treatment of inner ear diseases.

人类听力损失主要病理特征是毛细胞损伤，表现为永久性且不可逆，至今仍无有效治疗策略。因此，听力损失预防尤其重要。前期研究表明，非靶向性纳米粒可透过圆窗膜，并显著减弱顺铂在低频区（4, 8 kHz）引起的听力损失。然其对高频区（16, 24 kHz）听力下降无保护作用。因非靶向性纳米递送系统病变部位药物聚集能力有限，提示主动靶向纳米粒或能解决上述不足。本研究拟以特异靶向外毛细胞prestin蛋白的A666多肽为靶头，构建装载有模型药物地塞米松的PEG-PLA靶向纳米递送系统（A666-DEX-NP），并利用已成功构建的顺铂耳毒性豚鼠模型，探讨靶向纳米给药系统在预防顺铂耳毒性方面的优势及其作用机制。预实验结果表明，A666-DEX-NP在体内、外对外毛细胞具有较强的选择性。该项目将可与prestin特异性结合的多肽A666首次运用到耳蜗靶向给药的纳米递送系统中，为内耳相关疾病的治疗提供了新策略。

耳蜗外毛细胞损伤是导致顺铂引起的听力下降甚至丧失的关键原因。迄今为止，未有被FDA或SFDA批准上市的预防或治疗顺铂耳毒性的药物。本研究着重探讨新型多肽A666修饰，主动靶向外毛细胞药物递送系统作为“靶向定位”、“持续缓释”递送药物的可能性；以地塞米松（dexamathsone, DEX）为模型药物，并进一步以HEI-OC1和顺铂耳毒性豚鼠为体外细胞和体内动物模型，深入研究该主动靶向药物递送系统对顺铂耳毒性的预防作用及潜在作用机制。通过经典的巯基-氨基化学反应将A666多肽连接到纳米粒表面，XPS分析A666-NP纳米粒表面有0.4 %左右的S元素，而NP未检测到S元素，证实A666成功连接。最终获得A666-DEX-NP表面圆整光滑，平均粒径为157.80 ± 14.33 nm，表面电位为-32.53 ± 0.64 mV，包封率为1.20 ± 0.33 %，载药量为0.44 ± 0.12 %，体外人工淋巴液中缓释长达14 d。香豆素-6示踪A666-NP（A666-coumarin 6-NP） ，呈现时间依赖性HEI-OC1细胞内摄取特性；与prestin免疫荧光染色叠合证实A666-prestin相互作用摄取进入细胞。A666-coumarin 6-NP经圆窗膜给药1 h后，A666-NP“定位”聚集于外毛细胞；而无多肽修饰NP未观察到外毛细胞定位现象。细胞学实验表明，A666-DEX-NP在20，40，80 mg/ml浓度下显著拮抗顺铂（30 µM，24 h）细胞毒性；80 mg/ml浓度下有效抑制顺铂引起细胞凋亡。然而，相同浓度下DEX和DEX-NP并未表现出顺铂细胞毒性抑制和细胞凋亡拮抗作用。A666-DEX-NP经圆窗膜给药后，在内耳淋巴液中持续释放约48 h；而相同剂量的DEX，给药6 h后即被完全清除。由此，A666-DEX-NP体现出优良的“持续”累积释放药物，长时间维持有效药物浓度特性，这为DEX拮抗顺铂引起的听力下降提供了有力保障。基于此，顺铂腹腔注射前1 h，圆窗膜给于A666-DEX-NP，3 d后观察到在4 kHz，8 kHz和16 kHz频率下对豚鼠听力有显著的保护作用，本研究证实A666-DEX-NP具有良好的外毛细胞靶向特性，是内耳持续递送药物的理想载体。