Protein Labeling Facilitates the Understanding of Protein Corona Formation via Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy.

Once nanoparticles enter into the biological milieu, nanoparticle-biomacromolecule complexes, especially the protein corona, swiftly form, which cause obvious effects on the physicochemical properties of both nanoparticles and proteins. Here, the thermodynamic parameters of the interactions between water-soluble GSH-CdSe/ZnS core/shell quantum dots (GSH-QDs) and human serum albumin (HSA) were investigated with the aid of labeling fluorescence of HSA. It was proved that the labeling fluorescence originating from a fluorophore (BDP-CN for instance) could be used to investigate the interactions between QDs and HSA. Gel electrophoresis displayed that the binding ratio between HSA and QDs was ∼2:1 by direct visualization. Fluorescence resonance energy transfer (FRET) results indicated that the distance between the QDs and the fluorophore BDP-CN in HSA was 7.2 nm, which indicated that the distance from the fluorophore to the surface of the QDs was ∼4.8 nm. Fluorescence correlation spectroscopy (FCS) results showed that HSA formed a monolayer of a protein corona with a thickness of 5.5 nm. According to the spatial structure of HSA, we could speculate that the binding site of QDs was located at the side edge (not the triangular plane) of HSA with an equilateral triangular prism. The elaboration of the thermodynamic parameters, binding ratio, and interaction orientation will highly improve the fundamental understanding of the formation of protein corona. This work has guiding significance for the exploration of the interactions between proteins and nanomaterials.

New Insights on the Size-Dependent Inhibition of Enzymes by Gold Nanoparticles.

Particle size might affect the inhibition behaviors of gold nanoparticles (AuNPs) on enzyme activity by influencing the density of binding sites (ρ), the association constant (Ka), the steric hindrance of enzymes by AuNPs, the binding orientations of the enzyme on AuNPs, as well as the structural changes of enzymes. In previous studies, the effects of the above-mentioned factors, which could not be ignored in the applications of enzymatic electrochemistry, were often overshadowed by the effects of surface area. In order to study the size effect on the inhibition types and inhibitory ability of enzymes by AuNPs, we investigated the inhibition behaviors of chymotrypsin (ChT) by AuNPs with three different sizes (D1-AuNCs, D3-AuNPs, and D6-AuNPs) under the same surface area concentration. The results showed that both of the inhibition types and the inhibition ability varied with the particle size of AuNPs. D1-AuNCs inhibited ChT noncompetitively, while D3/D6-AuNPs inhibited ChT competitively. Contrary to the common sense, D6-AuNPs showed a weaker inhibitory ability than D3-AuNPs. By means of zeta potential, agarose gel electrophoresis, isothermal titration calorimetry, synchronous fluorescence spectroscopy, and circular dichroism, the mechanism of the weak inhibitory ability of D6-AuNPs was found to be the standing binding orientation caused by the small curvature. This work had certain guiding significance for the biosafety of AuNPs, the development of nanoinhibitors, as well as the applications of AuNPs in enzymatic electrochemistry.

Quantum Dots Meet Enzymes: Hydrophobicity of Surface Ligands and Size Do Matter.

Colloidal quantum dots (QDs) are a class of representative fluorescent nanomaterials with tunable, bright, and sharp fluorescent emission, with promising biomedical applications. However, their effects on biological systems are not fully elucidated. In this work, we investigated the interactions between QDs with different surface ligands and different particle sizes and α-chymotrypsin (ChT) from the thermodynamic and kinetic perspectives. Enzymatic activity experiments demonstrated that the catalytic activity of ChT was strongly inhibited by QDs coated with dihydrolipoic acid (DHLA-QDs) with noncompetitive inhibitions, whereas the QDs coated with glutathione (GSH-QDs) had weak effects. Furthermore, kinetics studies showed that different particle sizes of DHLA-QDs all had high suppressive effects on the catalytic activity of ChT. It was found that DHLA-QDs with larger particle sizes had stronger inhibition effects because more ChT molecules were bound onto the surface of QDs. This work highlights the importance of hydrophobic ligands and particle sizes of QDs, which should be considered as the primary influencing factors in the assessment of biosafety. Meanwhile, the results herein can also inspire the design of nano inhibitors.

Design and construction of competitive, noncompetitive and uncompetitive nano inhibitors of enzymes.

Three common types of reversible inhibitors, namely competitive, noncompetitive and uncompetitive inhibitors, were designed and constructed by using enzymes with different surface charges and gold nanoparticles with different surface ligands and particle sizes. To our knowledge, it is the first time that an uncompetitive nano inhibitor has been discovered.

Study on the Effects of Chinese Materia Medica Processing on the Hypoglycemic Activity and Chemical Composition of Anemarrhenae Rhizoma.

PURPOSE: To compare the hypoglycemic effects of different extracts of Anemarrhenae Rhizoma (AR) before and after being stir-baked with salt water on the diabetic mice and to detect the contents of 8 components in the corresponding active parts simultaneously using the UPLC-MS method, in order to screen the better extracts for diabetes and to clear the material basis for enhancing hypoglycemic activity of Anemarrhenae Rhizoma stir-baked with salt water (SAR). METHODS: Taking spontaneous type II diabetic db/db mice as models and fasting blood glucose (FBG), oral glucose tolerance test (OGTT), glycated hemoglobin or glycosylated hemoglobin (HbAlc), serum resistin (RESISTEIN), fasting insulin (FINS), superoxide dismutase (SOD), malondialdehyde (MDA), and nitric oxide (NO) as indicators, the hypoglycemic effects of different active parts of Anemarrhenae Rhizoma were evaluated. The chromatographic separation was performed on a Waters BEH C18 (2.1 mm × 50 mm, 1.7 µm) column using acetonitrile (B) and 0.1% formic acid in water (A) as mobile phases, and the flow rate was 0.3 ml/min. The column temperature was set as 28°C, and the injection volume was 10 µL. A mass spectrometer was connected to the UPLC system via an electrospray ionization (ESI) interface. Full-scan data acquisition was performed in the negative ion mode. RESULT: In the study of pharmacodynamics, the hypoglycemic effect of Anemarrhenae Rhizoma stir-baked with salt water is better than that of Anemarrhenae Rhizoma and the hypoglycemic effect of ethanol extract of Anemarrhenae Rhizoma is more remarkable than that of the decoction. The measured components all have a good linear relationship within their respective linear ranges (r ≥ 0.9990); the average recovery rates are 98.86%-100.69%, RSD <2.90%. Compared with the raw Anemarrhenae Rhizoma, the contents of Timosaponin AIII, Timosaponin BII, Timosaponin BIII, Anemarrhenasaponin I, Anemarrhenasaponin Ia, and Mangiferin of Anemarrhenae Rhizoma stir-baked with salt water are all higher, the changes of Timosaponin AI and Anemarrhenasaponin AII are not obvious, and all the contents of chemical composition in the ethanol extract of Anemarrhenae Rhizoma and Anemarrhenae Rhizoma stir-baked with salt water were obviously higher compared with the water decoction. CONCLUSION: The processing method, stir-baking with salt water, can increase the contents of active compositions in Anemarrhenae Rhizoma and strengthen the hypoglycemic effect. The ethanol extract of Anemarrhenae Rhizoma stir-baked with salt water is the better active site.