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

Area of Science:

• Biomaterials Science
• Chemical Engineering
• Molecular Biophysics

Background:

• Peptide and protein therapeutics face challenges in dosing due to rapid clearance and poor cell permeability.
• Nanocarrier encapsulation, like liposomes or polymersomes, offers a solution but faces manufacturing hurdles.
• Inverse Flash NanoPrecipitation (iFNP) enables production of highly loaded polymeric nanocarriers with proteins in a hydrophilic core.

Purpose of the Study:

• To investigate the conformational stability of proteins during iFNP processing.
• To characterize protein behavior in mixed-solvent environments encountered during iFNP.
• To assess the refolding capacity of proteins after encapsulation and release from nanocarriers.

Main Methods:

• Utilized explicit-solvent fully atomistic molecular dynamics simulations with enhanced sampling.
• Employed two-dimensional heteronuclear multiple-quantum coherence nuclear magnetic resonance spectroscopy (2D-HMQC NMR).
• Applied circular dichroism spectroscopy to monitor protein structure.
• Modeled interactions between water, dimethylsulfoxide (DMSO), and the Trp-cage protein TC5b.

Main Results:

• Protein unfolding was observed in high concentrations of DMSO (11 M) during simulated iFNP processing.
• NMR and circular dichroism confirmed structural changes consistent with unfolding.
• Complete refolding of the protein was demonstrated upon release from the nanocarrier into an aqueous environment.

Conclusions:

• Simulations and experimental techniques provide crucial insights into protein behavior during nanocarrier formulation.
• iFNP processing can induce temporary protein unfolding, but refolding is achievable.
• This study supports the viability of iFNP for formulating protein therapeutics in nanocarriers.