The clinical translation of protein drugs that act intracellularly is limited by the absence of safe and efficient intracellular protein delivery vehicles. Here, pH-sensitive coiled-coil peptide-cross-linked hyaluronic acid nanogels (HA-cNGs) were designed and investigated for targeted intracellular protein delivery to CD44 overexpressing MCF-7 breast cancer cells. HA-cNGs were obtained with a small size of 176 nm from an equivalent mixture of hyaluronic acid conjugates with GY(EIAALEK)₃GC (E3) and GY(KIAALKE)₃GC (K3) peptides, respectively, at pH 7.4 by nanoprecipitation. Circular dichroism (CD) proved the formation of coiled-coil structures between E3 and K3 peptides at pH 7.4 while fast uncoiling at pH 5.0. HA-cNGs showed facile loading of cytochrome C (CC) and greatly accelerated CC release under mild acidic conditions (18.4%, 76.8%, and 91.4% protein release in 24 h at pH 7.4, 6.0, and 5.0, respectively). Confocal microscopy and flow cytometry displayed efficient internalization of CC-loaded HA-cNGs and effective endosomal escape of CC in MCF-7 cancer cells. Remarkably, HA-cNGs loaded with saporin, a ribosome inactivating protein, exhibited significantly enhanced apoptotic activity to MCF-7 cells with a low IC₅₀ of 12.2 nM. These coiled-coil peptide-cross-linked hyaluronic acid nanogels have appeared as a simple and multifunctional platform for efficient intracellular protein delivery.

中文翻译：

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pH敏感的螺旋线圈肽交联的透明质酸纳米凝胶：合成和有针对性的细胞内蛋白质传递到CD44阳性癌细胞。

在细胞内起作用的蛋白质药物的临床翻译受到缺乏安全有效的细胞内蛋白质递送载体的限制。在这里，pH敏感的卷曲螺旋肽交联的透明质酸纳米凝胶（HA-cNGs）的设计和研究将靶向细胞内蛋白递送至过表达CD44的MCF-7乳腺癌细胞。从透明质酸结合物与GY（EIAALEK）₃ GC（E3）和GY（KIAALKE）₃的等效混合物中获得具有176 nm小尺寸的HA-cNGs通过纳米沉淀分别在pH 7.4的GC（K3）肽。圆二色性（CD）证明在pH 7.4的E3和K3肽之间形成卷曲螺旋结构，而在pH 5.0时快速解卷。HA-cNGs在轻度酸性条件下显示了细胞色素C（CC）的易装载性，并大大加速了CC的释放（在pH 7.4、6.0和5.0的24小时内分别释放了18.4％，76.8％和91.4％的蛋白质）。共聚焦显微镜和流式细胞仪显示MCF-7癌细胞中CC负载的HA-cNGs的有效内在化和CC的有效内体逸出。值得注意的是，载有saporin（一种核糖体失活蛋白）的HA-cNGs对MCF-7细胞的凋亡活性显着增强，IC ₅₀较低。为12.2 nM。这些卷曲螺旋肽交联的透明质酸纳米凝胶已显示为有效的细胞内蛋白递送的简单且多功能的平台。