脑胶质瘤是中枢神经系统中最常见的恶性肿瘤，复发率和死亡率较高。但由于 血脑屏障的存在，严重阻碍了药物对脑胶质瘤的治疗效果。靶向纳米药物递送系统 可将药物准确递送至脑部病灶部位，提高药物治疗效果，降低药物毒副作用。然而， 当纳米制剂进入生物环境时，通常会不可避免地与血浆蛋白发生相互作用，导致生 物分子会在其周围形成一个复杂的层状结构，这种层状结构被称为“蛋白冠（Protein Corona）”。蛋白冠的形成为原有的纳米制剂提供了新的生物特性，并介导了其与细 胞和生物屏障之间的相互作用。鉴于纳米制剂与生物环境之间的不可避免的作用， 我们将在纳米粒表面形成的蛋白冠视为主动靶向递送药物的工具，利用蛋白冠介导 脑胶质瘤的治疗。因此，本研究拟用 Aβ-CN 肽修饰的纳米胶束构建基于体内原位 载脂蛋白冠介导的紫杉醇纳米递送系统(PTX/Aβ-CN-PMs)， 用于靶向血脑屏障 （BBB）和脑胶质瘤表面高表达的低密度脂蛋白受体（LDLR）和低密度脂蛋白相关 蛋白 1（LRP1），从而高效地完成药物的脑靶向递送。Aβ-CN 肽是通过将 Aβ25-35 C 端蛋氨酸的羧酸结构改为酰胺形式而形成的，它不仅保留了与 Aβ 25-35 相似的特性 （例如有效结合载脂蛋白 E），而且还消除了 Aβ 25-35 的神经毒性，提高了其在体内 的生物安全性，这为 PTX/Aβ-CN-PMs 在体内安全的应用打下了坚实基础。

在本研究中，我们利用薄膜水化-超声法制备了 Aβ-CN 肽修饰的包载紫杉醇 （PTX）的 PEG-PLA 纳米胶束（PTX/Aβ-CN-PMs），旨在体内循环中 PTX/Aβ-CNPMs 表面的 Aβ-CN 肽可与载脂蛋白 E（ApoE）的脂质结合域结合，进而在 PTX/AβCN-PMs 周围特异性地形成富含 ApoE 的蛋白冠；而后，通过以正确取向暴露在胶 束表面的 ApoE 受体结合域主动靶向 BBB 和脑胶质瘤高表达的 LDLR 和 LRP1，有 效地介导药物脑靶向递送。通过 Western blot 和 LC-MS/MS 实验对 PTX/Aβ-CN-PMs 与小鼠血浆孵育后其形成的蛋白冠的种类和数量进行了表征，结果表明 Aβ-CN 肽 修饰可显著增加 PTX/Aβ-CN-PMs 表面蛋白冠中 ApoE 的含量。重要的是，体外和 体内的药效学研究实验表明，ApoE 蛋白冠的形成增强了纳米胶束对血脑肿瘤屏障 的渗透性；与游离紫杉醇相比，ApoE 蛋白冠介导的 PTX/Aβ-CN-PMs 可表现出更强 的抑制肿瘤细胞增殖和诱导肿瘤细胞凋亡的能力。另外，PTX/Aβ-CN-PMs 也展示 出了较好的抗胶质瘤效果和组织分布特征，可迅速积累于肿瘤组织中，与未形成 ApoE 蛋白冠的纳米胶束相比，有效延长了胶质瘤小鼠的中位生存期。总的来讲， 本课题将纳米制剂在体内遭遇到的不利现象（蛋白冠）转化为脑主动靶向递送的关 键，为利用体内原位形成的蛋白冠介导的主动靶向药物递送系统的设计提供了思路 和策略，促进了主动靶向药物递送策略的发展。

Glioma, as one of the most common malignant tumors in the central nervous system, usually has high recurrence and mortality rates. Blood-brain barrier severely hinders the therapeutic efficacy of drugs in glioma. With the development of nanotechnology, the targeted nanomedicine delivery systems have provided an effective approach to accurately deliver drugs to the brain lesion site, improve the therapeutic effect, and reduce toxic side effects. However, after entering the biological environment, targeted nanoformulations usually inevitably interact with plasma proteins in vivo, resulting in the formation of a complex layer of biomolecules around them, known as "protein corona". It provides new biological properties to the original nanoformulations and mediates the interactions with cells and biological barriers. Given the inevitable interactions, we regard nanoparticleprotein interactions as a tool for designing protein corona-mediated drug delivery systems. Herein, we demonstrate the successful application of protein corona-mediated braintargeted nanomicelles in the treatment of glioma, loading them with paclitaxel (PTX), and decorating them with amyloid β-protein (Aβ)-CN peptide (PTX/Aβ-CN-PMs) for targeting low-density lipoprotein receptor (LDLR) and low-density lipoprotein-related protein 1 (LRP1) expressed on BBB and gliomas. The Aβ-CN peptide is formed by changing the carboxylic acid structure of the C-terminal methionine of Aβ 25-35 to an amide form, which not only maintains similar properties to those of Aβ25-35 (e.g., efficient binding of ApoE), but also eliminates the neurotoxicity of Aβ25-35 and improves its biosafety in vivo, laying a good foundation for the safe application of PTX/Aβ-CN-PMs in vivo.

Therefore, in this study, we prepared Aβ-CN peptide-modified PEG-PLA nanomicelles (PTX/Aβ-CN-PMs) encapsulated with PTX using a thin-film hydrationsonication method, which aimed that the Aβ-CN peptide on the surface of PTX/Aβ-CNPMs could bind to the lipid binding site of ApoE during in vivo circulation and specifically form ApoE-enriched protein corona around PTX/Aβ-CN-PMs. Then, the receptor binding domains of ApoE exposed on the surface of nanomicelles in the correct orientation can actively target LDLR and LRP1 highly expressed on the BBB and glioma, effectively mediating drug brain-targeted delivery. The specific formation of the ApoE-enriched protein corona around nanomicelles was characterized by Western blot analysis and LCMS/MS. The results showed that Aβ-CN peptide modification significantly increased the ApoE content in protein corona formed on the surface of PTX/Aβ-CN-PMs. In vitro and in vivo studies showed that the formation of the ApoE-enriched protein corona 

significantly enhanced permeability to the blood–brain tumor barrier in vitro. In addition, PTX/Aβ-CN-PMs with ApoE-enriched protein corona had a greater ability to inhibit cell proliferation and induce cell apoptosis than Taxol. Importantly, PTX/Aβ-CN-PMs exhibited better anti-glioma effects and tissue distribution profile with rapid accumulation in glioma tissues in vivo and prolonged median survival in glioma-bearing mice compared to PMs without the ApoE protein corona.

In conclusion, this research demonstrated that the unfavorable phenomenon (protein corona) encountered in vivo by targeted nanoformulations can be converted into a key approach for active targeting brain delivery, which provides insights and strategies for the design of active targeting drug delivery systems mediated by in situ formation of protein corona in vivo and promotes the development of drug delivery.