非膜电位依赖型线粒体靶向传递策略：治疗药物性耳聋

Certain clinically useful and essential pharmacological therapies may result in temporary or permanent hearing loss due to interactions of the drugs and cells of the inner ear. More than 70% cases of hearing loss in China are attributable to ototoxicity. However, the treatment of it is especially lack. Hearing loss due to ototoxicity is the result of apoptosis. In detail, the outer hair cells of the basal turn of the cochlea are highly sensitive to the drugs and seem to die from apoptosis. The mitochondria within human cells play a major role in a variety of critical processes involved in cell survival and death. Mitochondrial dysfunction is an acute or chronic disturbance in metabolism, bioenergetics, or signaling pathways that occur within mitochondria, which can give rise to a plethora of diseases with variable symptoms including hearing loss. That is to say, the mitochondrion is a major regulator of cell death pathway such as apoptosis. Clinical trials with mitochondria acting therapeutics have failed to show benefits in humans owing to the logistics of safe drug delivery to the mitochondria in sufficient amounts and the toxicity associated with high doses. Although several substances are known to act on mitochondria, their dependence on the membrane potential in mitochondria also constitutes a major drawback. As increasing numbers of lipophilic cations (such as TPP) enter the organelle, the potential gradients diminish and lead to mitophage. Furthermore, the uptake of these vectors is self-limiting due to the inevitable depolarization of mitochondria. A novel class of small cell permeable peptide antioxidants that target mitochondria in a potential independent manner were reported recently. The Szeto-Schiller (SS) peptides do not cause mitochondrial depolarization even at 1 mM. Thus, SS antioxidants peptides represent a novel approach, with targeted delivery of vector to the mitochondria. The potential of nanoparticles to improve any therapy lies in their ability to deliver payloads directly to the cells of interest and simultaneously enhance stability and pharmacokinetics. An equally important consideration is whether the same nanoparticles can deliver the therapeutic payload to the intended target inside the cells. Delivery of payloads using biodegradable and FDA approved PLGA based nanoparticles to the target sites has been well characterized, but the intracellular barriers that delivery carriers must overcome to reach subcellular targets are less well understood. In this project, we describe an engineering approach to the design and execution of a polymer-blending technology to fine-turn size and surface charges of the resultant nanoparticles to provide an optimized PLGA-based nanoparticles platform for mitochondria targeted delivery of therapeutics. We postulate that pharmaceutical interventions via mitochondria targeted therapy that inhibit apoptosis of hair cells will prevent the hearing loss due to ototoxicity.

药物致聋是听力障碍的主要病因，而相关治疗研究却极为匮乏。药物致聋的关键环节是线粒体损伤导致的内耳毛细胞凋亡。本课题拟构建SS四肽修饰的PLGA线粒体靶向纳米粒，在不影响线粒体膜功能的同时，通过引导治疗分子递送入线粒体，逆转毛细胞凋亡以实现治疗目的。SS四肽是非依赖于线粒体膜电位的靶向序列，与常规线粒体靶向单元相比，这种新型的线粒体靶向分子不会诱发线粒体膜电位的耗散，有利于其膜结构的维持，这对以抑制细胞凋亡为目的的治疗十分关键。在内耳给药传递系统的研究工作基础上，申请者着重研究以下三个方面，来为药致耳聋治疗制剂的开发提供理论基础：①SS四肽靶向纳米粒理化性质对毛细胞摄取、线粒体靶向效率的影响；②靶向纳米粒的内涵体和溶酶体逃逸功能和机制分析；③从抑制凋亡信号、上调毛细胞内在保护机制和缓解氧化应激三个方面探讨所构建体系对逆转氨基糖甙类药物所致毛细胞凋亡的机制。

药物性耳聋已成为影响人类健康的重要问题，临床上对其尚无行之有效的治疗策略。氨基糖苷类抗生素引起的听力障碍主要由内耳机械感觉毛细胞的永久性损伤造成。近年来，越来越多的研究表明线粒体损伤是内耳毛细胞凋亡的关键因素，而线粒体靶向递药为增加治疗药物疗效、降低相关毒性提供了重要手段。.为验证线粒体靶向的基本假说，本项目首先选取线粒体内膜电位驱动的TPP为靶向序列修饰PLGA纳米粒，以期达到较高的对抗庆大霉素致毒活性。结果表明，载药PLGA-b-PEG-TPP NPs能够实现保护内耳重要感觉细胞—毛细胞的作用，特别是在慢毒模式下，可将毛细胞存活率从15%提高至70%，提示TPP修饰的纳米粒具有改善药物胞内分布、增强药物对抗庆大霉素致毒的潜力，但同时我们还发现TPP修饰的NPs具有一定的毛细胞毒性，提示膜电位依赖型线粒体靶向策略可能不适用于药物性耳聋的治疗。.基于此，课题选取非膜电位依赖的线粒体靶向小肽SS31修饰PLGA纳米粒，结果发现， SS31引导的线粒体靶向PLGA纳米载药系统可改善在急毒和慢毒作用模式下毛细胞存活率，提示该递送系统在抵抗庆大霉素毒性、保护毛细胞结构完整性方面具有一定的应用潜力。.由于SS31引导的线粒体靶向PLGA纳米载药系统并未实现非膜电位依赖型的线粒体靶向，因此，我们通过对SS类似物的构效分析，最终优选FF-1 (D-Arg-Dmt-Orn-Phe-NH2)和 RF-2(D-Arg-Dmt-Arg-Phe-NH2)用于后期实验。值得注意的是，FF-1引导的纳米粒（FF-1-PLGA-NPs）靶向线粒体不依赖于其膜电位，相反， RF-2-PLGA-NPs靶向高度依赖线粒体膜电位。FF-1-PLGA-NPs可有效地保护毛细胞免受庆大霉素诱导的损伤，并进一步拓宽了载药系统的作用窗口。.值得关注的是，我们发现PLGA纳米粒抑制了毛细胞纤毛上的力传导通道（MET channel），尽管经实验证明这种MET通道的抑制作用是一过性的，即清除后通道活性可恢复，但提示PLGA纳米粒内耳摄取后可能影响听觉电生理，因此我们后续选用了成药性较好的脂质体进行了相关实验，发现脂质体对MET通道的活性无干扰，是一种值得开发、用于内耳给药的递药系统，经SS31修饰后的载药系统改善了米诺环素对抗药物性耳聋的活性。