Protein Nanocage as a pH-Switchable Pickering Emulsifier

Encapsulation of active compounds in Pickering emulsions using bioderived protein-based stabilizers holds potential for the development of novel formulations in the fields of foods and cosmetics. We employ a dodecahedron hollow protein nanocage as a pH-switchable Pickering emulsifier. E2 protein nanocages are derived from pyruvate dehydrogenase multienzyme complex from <i>Geobacillus stearothermophilus</i> which adsorb at the oil/water interface at neutral and basic pH’s and stabilize the Pickering emulsions, while in the acidic range, at pH ∼4, the emulsion separates into emulsion and serum phases due to flocculation. The observed process is reversible for at least five cycles. Optimal formulation of a Pickering emulsion composed of rosemary oil, an essential oil, and water has been achieved by ultrasonication and results in droplets of approximately 300 nm in diameter with an oil/water ratio of 0.11 (v/v) and 0.30–0.35% (wt %). Ionic stabilization is observed for concentrations up to 250 mM NaCl and pH values from 7 to 11. The emulsions are stable for at least 10 days when stored at different temperatures up to 50 °C. The resulting Pickering emulsions of different compositions also form a gel-like structure and show shear thinning behavior under shear stress at a higher oil/water ratio

The E2 porous protein cage.

<p>(A) and (B) E2 protein cage three-dimensional structure (adapted from PDB ID: 1b5s).[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162848#pone.0162848.ref042" target="_blank">42</a>] The inserts show typical electron microscopy images of the 5-fold axis (A) and 2-fold axis (B) orientations of the protein cage E2. The diameter <i>D</i> of the E2 protein cage is 25 nm. The diameter <i>d</i> of each pore is 6 nm. (C) E2 protein cage inner surface presenting RDGE loop sequences in blue. The diameter δ of the cage core is 13 nm.</p

Transmission electron microscopy of samples of Ni-NTA-functionalised gold nanoparticles and E2 protein cages.

<p>Ni-NTA-functionalised gold nanoparticles mixed with (A) E2-WT, (B) E2-LH2, (C) E2-LH5, (D) E2-LH6. White circles indicate Ni-NTA-functionalised gold nanoparticles internalised into oligohistidine modified E2 protein cages. The samples were stained with 1% (w/v) phosphotungstic acid. Scale bars are 100 nm.</p

Electron microscopy of wild-type and engineered E2 protein cages.

<p>Images of the E2 protein cages: (A, E) E2-WT; (B, F) E2-LH2; (C, G) E2-LH5; (D, H) E2-LH6. (A–D) scale bars are 100 nm. (E–H) scale bars are 50 nm.</p

Dynamic light scattering analysis of wild-type and engineered E2 protein cages with different oligohistidine sequences at the RDGE loop.

<p>Dynamic light scattering analysis of wild-type and engineered E2 protein cages with different oligohistidine sequences at the RDGE loop.</p

Oligonucleotides for plasmid construction.

<p>Oligonucleotides for plasmid construction.</p

Method for specific internalisation of peptide coated gold nanoparticles into engineered E2 protein cages.

<p>(a) Site-directed mutagenesis of E2 protein cage’s core with oligohistidine sequences (blue) at the RDGE loop (green) on E2 protein subunits; (b) Surface coating of 3.9 nm gold nanoparticles with a self-assembled monolayer made of peptidols and thiolated alkane ethylene glycol (EG) ligands, functionalised with Ni²⁺ nitrilotriacetic moieties (NTA, Ni²⁺); (c) Specific internalisation by affinity binding of Ni-NTA-functionalised peptide coated gold nanoparticles into E2 protein cages presenting oligohistidine sequences.</p

Ni-NTA-functionalised peptide coated gold nanoparticles.

<p>(A) Size distribution of 212 gold nanoparticles with an average diameter of 3.9 ± 0.8 nm. The insert shows a typical electron microscopy image used for size measurement. Immobilisation of Ni-NTA-functionalised peptide coated gold nanoparticles on a hexa-histidine loaded resin. (B) NTA-functionalised SAM coated gold nanoparticles not loaded with Ni²⁺ present no non-specific binding to hexa-histidine resin. (C) 10% (mol:mol) Ni-NTA-functionalised peptide coated gold nanoparticles fully bind to hexa-histidine resin as no free gold nanoparticles are found in the clear supernatant.</p

Micelle/Silica Co-protected Conjugated Polymer Nanoparticles for Two-Photon Excited Brain Vascular Imaging

Large two-photon absorption cross section and high fluorescence quantum yield (QY) of a fluorescent probe is highly desirable to achieve high resolution in two-photon excited fluorescence imaging. Taking poly­(9,9-dihexylfluorene-<i>alt</i>-2,1,3-benzothiadiazole) (PFBT) as an example, we report a one-step approach to synthesize PFBT loaded nanoparticles (NPs) with both large two-photon absorption cross section and high fluorescence QY in aqueous media through a micelle and silica coprotection strategy. The PFBT loaded NPs show a two-photon absorption cross section of 1085 GM at 810 nm based on polymer chain concentration and an emission maximum at 545 nm with a high fluorescence QY of 75%. The fluorescence lifetime investigation reveals that the high fluorescence QY is mainly due to reduced polymer aggregation and minimized environment influence on conjugated polymer (CP) fluorescence quenching. The synthesized PFBT NPs have shown good colloid stability and photostability as well as benign biocompatibility, which have been further applied to visualize the mouse brain vasculature through intravital two-photon excited brain vascular imaging with high contrast. The developed micelle/silica coprotection strategy should be generally applicable to other CP NPs with improved brightness and stability for various biological applications

Precise and Long-Term Tracking of Adipose-Derived Stem Cells and Their Regenerative Capacity <i>via</i> Superb Bright and Stable Organic Nanodots

Monitoring and understanding long-term fate and regenerative therapy of administrated stem cells <i>in vivo</i> is of great importance. Herein we report organic nanodots with aggregation-induced emission characteristics (AIE dots) for long-term tracking of adipose-derived stem cells (ADSCs) and their regenerative capacity in living mice. The AIE dots possess high fluorescence (with a high quantum yield of 25 ± 1%), excellent biological and photophysical stabilities, low <i>in vivo</i> toxicity, and superb retention in living ADSCs with negligible interference on their pluripotency and secretome. These AIE dots also exhibit superior <i>in vitro</i> cell tracking capability compared to the most popular commercial cell trackers, PKH26 and Qtracker 655. <i>In vivo</i> quantitative studies with bioluminescence and GFP labeling as the controls reveal that the AIE dots can precisely and quantitatively report the fate of ADSCs and their regenerative capacity for 42 days in an ischemic hind limb bearing mouse model