4.8 Article

Stability of Protein Structure during Nanocarrier Encapsulation: Insights on Solvent Effects from Simulations and Spectroscopic Analysis

期刊

ACS NANO
卷 14, 期 12, 页码 16962-16972

出版社

AMER CHEMICAL SOC
DOI: 10.1021/acsnano.0c06056

关键词

molecular dynamics; nanocarrier; protein delivery; simulations; spectroscopy

资金

  1. Princeton Center for Complex Materials, a National Science Foundation (NSF)-MRSEC program [DMR-1420541]
  2. National Science Foundation [ACI-1548562]
  3. Princeton Institute for Computational Science and Engineering (PICSciE)
  4. Office of Information Technology's High Performance Computing Center and Visualization Laboratory at Princeton University
  5. Optimeos Life Sciences
  6. PhRMA Foundation Pre-Doctoral Fellowship in Pharmaceutics
  7. NSF [MCB-1409402, MCB-1947720]

向作者/读者索取更多资源

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 TCSb 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 TCSb 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.

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