Abstract Ferritin, a widely expressed iron binding protein, has emerged as a promising drug delivery vehicle due to its unique architecture, intrinsic tumor targeting property, and excellent biocompatibility. However, the translation studies of ferritin drug carrier are impeded by the low efficiency and low yield of the drug loading process, which typically employed Urea and pH dependent dis-assembly/reassembly methods. Here, based on crystal structure and protein mutation analyses, we identified a natural drug entry channel existed on the shell of recombinant human H-ferritin (HFn). Moreover, the opening state of this drug entry channel is sensitive to temperature changes. Based on these findings, we develop a simple channel-based drug loading strategy that avoids the denaturation of HFn protein cage with denaturing agents. Loading doxorubicin (Dox) into the HFn protein cage (HFn-Dox) by the channel-based strategy yields significantly higher drug loading efficiency, increased HFn recovery rate, and better stability than that of the denaturation-based methods. Importantly, animal experiments showed that the channel-loaded HFn-Dox had excellent biosafety and significantly improved antitumor activity when compared with the HFn-Dox prepared by the denaturation-based methods. We also found that the drug entry channel appears unique for HFn protein cage and is accessible to multiple small molecule anticancer drugs. Thus, the drug entry channel of HFn protein cage identified in this study provides a novel and highly effective drug loading approach for preparing HFn-drug formulations, which will greatly facilitate the translational study of ferritin drug carriers in cancer-targeting therapies.

中文翻译：

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铁蛋白纳米笼中的天然药物进入通道

摘要 铁蛋白是一种广泛表达的铁结合蛋白，由于其独特的结构、内在的肿瘤靶向特性和优异的生物相容性，已成为一种有前途的药物递送载体。然而，铁蛋白药物载体的翻译研究受到载药过程的低效率和低产率的阻碍，该过程通常采用尿素和 pH 依赖性分解/重组方法。在这里，基于晶体结构和蛋白质突变分析，我们确定了重组人 H-铁蛋白 (HFn) 外壳上存在的天然药物进入通道。而且，这个药物进入通道的开放状态对温度变化很敏感。基于这些发现，我们开发了一种简单的基于通道的药物加载策略，避免了 HFn 蛋白笼与变性剂的变性。与基于变性的方法相比，通过基于通道的策略将阿霉素 (Dox) 加载到 HFn 蛋白笼 (HFn-Dox) 中可产生显着更高的载药效率、增加的 HFn 回收率和更好的稳定性。重要的是，动物实验表明，与通过基于变性方法制备的 HFn-Dox 相比，通道负载的 HFn-Dox 具有出色的生物安全性和显着提高的抗肿瘤活性。我们还发现药物进入通道对于 HFn 蛋白笼来说似乎是独一无二的，并且可用于多种小分子抗癌药物。因此，本研究中鉴定的 HFn 蛋白笼的药物进入通道为制备 HFn 药物制剂提供了一种新颖且高效的载药方法，