Vallerinteavide Mavelli Girish, Sadeghi Samira, Vaidya Siddhesh Sujit, Kong Shik Nie, Drum Chester Lee
Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, Singapore 117599, Singapore.
Genome Institute of Singapore, Agency for Science, Technology and Research (A*Star), Singapore 138672, Singapore.
Pharmaceutics. 2021 Oct 26;13(11):1790. doi: 10.3390/pharmaceutics13111790.
Protein macromolecules occur naturally at the nanoscale. The use of a dedicated nanoparticle as a lyophilization excipient, however, has not been reported. Because biopolymeric and lipid nanoparticles often denature protein macromolecules and commonly lack the structural rigidity to survive the freeze-drying process, we hypothesized that surrounding an individual protein substrate with a nanoscale, thermostable exoshell (tES) would prevent aggregation and protect the substrate from denaturation during freezing, sublimation, and storage. We systematically investigated the properties of tES, including secondary structure and its homogeneity, throughout the process of lyophilization and found that tES have a near 100% recovery following aqueous reconstitution. We then tested the hypothesis that tES could encapsulate a model substrate, horseradish peroxidase (HRP), using charge complementation and pH-mediated controlled assembly. HRP were encapsulated within the 8 nm internal tES aqueous cavity using a simplified loading procedure. Time-course experiments demonstrated that unprotected HRP loses 95% of activity after 1 month of lyophilized storage. After encapsulation within tES nanoparticles, 70% of HRP activity was recovered, representing a 14-fold improvement and this effect was reproducible across a range of storage temperatures. To our knowledge, these results represent the first reported use of nanoparticle encapsulation to stabilize a functional macromolecule during lyophilization. Thermostable nanoencapsulation may be a useful method for the long-term storage of labile proteins.
蛋白质大分子天然存在于纳米尺度。然而，尚未有将专用纳米颗粒用作冻干辅料的报道。由于生物聚合物和脂质纳米颗粒常常会使蛋白质大分子变性，且通常缺乏在冻干过程中存活所需的结构刚性，我们推测用纳米级的热稳定外壳（tES）包裹单个蛋白质底物能够防止聚集，并在冷冻、升华和储存过程中保护底物不发生变性。我们在整个冻干过程中系统地研究了tES的性质，包括二级结构及其均匀性，发现tES在水相重构后回收率接近100%。然后，我们利用电荷互补和pH介导的可控组装，测试了tES能否包裹模型底物辣根过氧化物酶（HRP）这一假设。通过简化的加载程序，将HRP包裹在8纳米的内部tES水腔中。时间进程实验表明，未受保护的HRP在冻干储存1个月后丧失95%的活性。在包裹于tES纳米颗粒后，70%的HRP活性得以恢复，提高了14倍，且这种效果在一系列储存温度下均可重现。据我们所知，这些结果代表了首次报道使用纳米颗粒包裹来在冻干过程中稳定功能性大分子。热稳定纳米包裹可能是一种用于长期储存不稳定蛋白质的有用方法。
