An official website of the United States government
Many biological and biotechnological processes are controlled by protein–protein and protein–solvent interactions. In order to understand, predict, and optimize such processes, it is important to understand how solvents affect protein structure during protein–solvent interactions. In this study, all-atom molecular dynamics are used to investigate the structural dynamics and energetic properties of a C-terminal domain of the Rift Valley Fever Virus L protein solvated in glycerol and aqueous glycerol solutions in different concentrations by molecular weight. The Generalized Amber Force Field is modified by including restrained electrostatic potential atomic charges for the glycerol molecules. The peptide is considered in detail by monitoring properties like the root-mean-squared deviation, root-mean-squared fluctuation, radius of gyration, hydrodynamic radius, end-to-end distance, solvent-accessible surface area, intra-potential energy, and solvent–peptide interaction energies for hundreds of nanoseconds. Secondary structure analysis is also performed to examine the extent of conformational drift for the individual helices and sheets. We predict that the peptide helices and sheets are maintained only when the modeling strategy considers the solvent with lower glycerol concentration. We also find that the solvent-peptide becomes more cohesive with decreasing glycerol concentrations. The density and radial distribution function of glycerol solvent calculated when modeled with the modified atomic charges show a very good agreement with experimental results and other simulations at 298.15K.
more »
« less
Stability of Protein Structure during Nanocarrier Encapsulation: Insights on Solvent Effects from Simulations and Spectroscopic Analysis
ABSTRACT: 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.
more »
« less
- Award ID(s):
- 1947720
- PAR ID:
- 10205659
- Date Published:
- Journal Name:
- ACS Nano
- ISSN:
- 1936-0851
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Betulin is a promising natural organic substance due to its antibacterial, fungicidal, and antitumor properties, as are their derivatives. The particle size of betulin can reach several tens of micrometers, and its thickness is several microns. There are various ways of processing betulin, but the most promising are solution methods (applying thin layers, impregnation, etc.). Application or impregnation of various materials is carried out using betulin; however, currently known solvents do not allow obtaining solutions with the necessary content of it. Since a number of direct solvents are already known for betulin, which provides only low-concentration solutions, the use of complex systems based on two solvents can become the optimal solution to the problem. The literature data show that the use of mixtures of solvents allows for the preparation of homogeneous solutions, for example, for natural polymers like cellulose, etc. This approach to obtaining solutions has become the basis for the processing of betulin. The use of a mixed solvent based on ethanol and DMSO for the preparation of betulin solutions has been proposed for the first time. The solubility of betulin in a mixture system with a ratio of components of 50 wt.% to 50 wt.% was studied, and a solubility curve was plotted. It is shown that the use of a two-component solvent makes it possible to transfer up to 10% of betulin into solution, which is almost twice as much as compared to already known solvents. The rheological properties of the obtained solutions have been studied. The viscosity of betulin solutions in a complex solvent depends on its content and temperature, so for 7% solutions at 70 °C, it is approximately 0.008 Pa*s. Applying betulin to the surface of the cardboard increases its hydrophobic properties and repellency.more » « less
-
null (Ed.)Polymeric membranes for separation of pharmaceutical intermediates/products by organic solvent nanofiltration (OSN) have to be highly resistant to many organic solvents including high-boiling polar aprotic ones, e.g., N- methyl-2-pyrollidone (NMP), dimethylsulfoxide (DMSO), dimethylformamide (DMF). Unless cross-linked, few polymers resist swelling or dissolution in such solvents; however particular perfluoropolymers are resistant to almost all solvents except perfluorosolvents. One such polymer, designated AHP1, a glassy amorphous hydrophobic perfluorinated polymer, has been studied here. Additional perfluoropolymers studied here are hydrophilically modified (HMP2 and HMP3) versions to enhance the flux of polar aprotic solvents. OSN performances of three types of membranes including the hydrophilically modified ones were studied via solvent flux and solute rejection at pressures up to 5000 kPa. The solutes were four active pharmaceutical ingredients (APIs) or pharmaceutical intermediates having molecular weights (MWs) between 432 and 809 Da and three dyes, Oil Blue N (378 Da), Sudan Black B (456 Da), Brilliant Blue R (826 Da). Solvents used were: ethyl acetate, toluene, n- heptane, iso-octane, DMSO, tetrahydrofuran (THF), DMF, acetone, NMP, methanol. Test cells included stirred cells and tangential flow cells. Pure solvent fluxes through three membrane types were characterized using a particular parameter employing various solvent properties. All three membranes achieved high solute rejections around 91–98% at ambient temperatures. HMP2 membrane achieved 95% solute rejection for an API (809 Da) in DMSO at a high temperature, 75 ◦C. A two-stage simulated nanofiltration process achieved 99%+ rejection of a pharmaceutical intermediate (MW, 432 Da) in 75v% NMP-25v% ethyl acetate solution.more » « less
-
Neonatal hypoxic-ischemic encephalopathy is the leading cause of permanent brain injury in term newborns and currently has no cure. Catalase, an antioxidant enzyme, is a promising therapeutic due to its ability to scavenge toxic reactive oxygen species and improve tissue oxygen status. However, upon in vivo administration, catalase is subject to a short half-life, rapid proteolytic degradation, immunogenicity, and an inability to penetrate the brain. Polymeric nanoparticles can improve pharmacokinetic properties of therapeutic cargo, although encapsulation of large proteins has been challenging. In this paper, we investigated hydrophobic ion pairing as a technique for increasing the hydrophobicity of catalase and driving its subsequent loading into a poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticle. We found improved formation of catalase-hydrophobic ion complexes with dextran sulfate (DS) compared to sodium dodecyl sulfate (SDS) or taurocholic acid (TA). Molecular dynamics simulations in a model system demonstrated retention of native protein structure after complexation with DS, but not SDS or TA. Using DS-catalase complexes, we developed catalase-loaded PLGA-PEG nanoparticles and evaluated their efficacy in the Vannucci model of unilateral hypoxic-ischemic brain injury in postnatal day 10 rats. Catalase-loaded nanoparticles retained enzymatic activity for at least 24 h in serum-like conditions, distributed through injured brain tissue, and delivered a significant neuroprotective effect compared to saline and blank nanoparticle controls. These results encourage further investigation of catalase and PLGA-PEG nanoparticle-mediated drug delivery for the treatment of neonatal brain injury. View Full-Textmore » « less
-
To stabilize and transport them through complex systems, nanoparticles are often encapsulated in polymeric nanocarriers, which are tailored to specific environments. For example, a hydrophilic polymer capsule maintains circulation and stability of nanoparticles in aqueous environments. A more highly-designed nanocarrier might have a hydrophobic core and a hydrophilic shell to allow transport of hydrophobic nanoparticles and pharmaceuticals through physiological media. Polydimethylsiloxane, PDMS, is a hydrophobic material in a liquidlike state at room temperature. The preparation of stable, aqueous dispersions of PDMS droplets in water is problematic due to the intense mismatch in surface energies between PDMS and water. The present work describes the encapsulation of hydrophobic metal- and metal oxide nanoparticles within PDMS nanodroplets using flash nanoprecipitation. The PDMS is terminated by amino groups and the nanodroplet is capped with a layer of poly(styrene sulfonate), forming a glassy outer shell. The hydrophobic nanoparticles nucleate PDMS droplet formation, decreasing the droplet size. The resulting nanocomposite nanodroplets are stable in aqueous salt solutions without the use of surfactants. The hierarchical structuring, elucidated with small angle x-ray scattering, offers a new platform for the isolation and transport of hydrophobic molecules and nanoparticles through aqueous systems.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1021/acsnano.0c06056
