Stability of Protein Structure during Nanocarrier Encapsulation: Insights on Solvent Effects from Simulations and Spectroscopic Analysis
Stability of Protein Structure during Nanocarrier Encapsulation: Insights on Solvent Effects from Simulations and Spectroscopic Analysis
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纳米载体封装过程中蛋白质结构的稳定性：从模拟和光谱分析中了解溶剂效应
DOI：
10.1021/acsnano.0c06056
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发表时间：
2020
期刊：
影响因子：
17.1
通讯作者：
Debenedetti, Pablo G.
中科院分区：
文献类型：
--
作者：
Markwalter, Chester E.;Uralcan, Betul;Pelczer, István;Zarzhitsky, Shlomo;Hecht, Michael H.;Prud’homme, Robert K.;Debenedetti, Pablo G.
肽和蛋白质治疗剂的给药由于从血池中的快速清除和差的细胞膜渗透性而变得复杂。长期以来，人们一直在探索包封到纳米载体如脂质体或聚合物囊泡中以克服这些限制，但制造挑战限制了这些方法的临床转化。最近，已经开发了反相快速纳米沉淀（iFNP）以生产高度负载的聚合物纳米载体，其具有包含在亲水性核内的肽或蛋白质，通过疏水性聚合物壳稳定。封装蛋白质与高阶结构需要了解如何处理可能会影响他们的构象状态。我们展示了一种结合实验/模拟的方法来表征蛋白质的行为，在iFNP处理步骤中使用色氨酸笼蛋白TC 5 b作为模型。显式溶剂全原子分子动力学模拟与增强的采样技术，再加上二维异质结多量子相干核磁共振光谱（2D-HMQC NMR）和圆二色性，以确定在混合溶剂暴露过程中遇到的iFNP处理的TC 5 b的结构。模拟涉及混合溶剂和蛋白质的原子模型，以捕捉水，二甲基亚砜（DMSO）和蛋白质之间的氢键和疏水相互作用的复杂性。组合分析揭示了蛋白质在11 M DMSO中的结构解折叠，但证实了从聚合物纳米载体释放回到水相中后的完全重折叠。这些结果突出了模拟和NMR为纳米载体中蛋白质的配制提供的见解。
The dosing of peptide and protein therapeutics is complicated by rapid clearance from the blood pool and poor cellular membrane permeability. Encapsulation into nanocarriers such as liposomes or polymersomes has long been explored to overcome these limitations, but manufacturing challenges have limited clinical translation by these approaches. Recently, inverse Flash NanoPrecipitation (iFNP) has been developed to produce highly loaded polymeric nanocarriers with the peptide or protein contained within a hydrophilic core, stabilized by a hydrophobic polymer shell. Encapsulation of proteins with higher-order structure requires understanding how processing may affect their conformational state. We demonstrate a combined experimental/simulation approach to characterize protein behavior during iFNP processing steps using the Trp-cage protein TC5b as a model. Explicit-solvent fully atomistic molecular dynamics simulations with enhanced sampling techniques are coupled with two-dimensional heteronuclear multiple-quantum coherence nuclear magnetic resonance spectroscopy (2D-HMQC NMR) and circular dichroism to determine the structure of TC5b during mixed-solvent exposure encountered in iFNP processing. The simulations involve atomistic models of mixed solvents and protein to capture the complexity of the hydrogen bonding and hydrophobic interactions between water, dimethylsulfoxide (DMSO), and the protein. The combined analyses reveal structural unfolding of the protein in 11 M DMSO but confirm complete refolding after release from the polymeric nanocarrier back into an aqueous phase. These results highlight the insights that simulations and NMR provide for the formulation of proteins in nanocarriers.