Controlled Nutrient Delivery through a pH-Responsive Wood Vehicle.

To lower the risk of disease and improve health, many nutrients benefit from intestinal-targeted delivery. Here, we present a nutrient-delivery system based on a pH-responsive "wood scroll", in which nutrients are stored, protected, and controllably released through the rolled structure and natural microchannels of a flexible wood substrate, thus ensuring higher bioactivity as well as prolonged steady release of the nutrient load to the intestine. We loaded the wood's natural microchannels with probiotics as a proof-of-concept demonstration. The probiotic-loaded wood scrolls can survive the simulated conditions of the stomach with a high survival rate (95.40%) and exhibit prolonged release (8 h) of the probiotic load at a constant release rate (4.17 × 108 CFUs/h) in the simulated conditions of the intestine. Moreover, by modifying the macroscopic geometry and microstructures of the wood scrolls, both the nutrient loading and release behaviors can be tuned over a wide range for customized or personalized nutrient management. The wood scrolls can also deliver other types of nutrients, as we demonstrate for tea polyphenols and rapeseed oil. This wood scroll design illustrates a promising structurally controlled strategy for the delivery of enteric nutrients using readily available, low-cost, and biocompatible biomass materials that have a naturally porous structure for nutrient storage, protection, and controlled release.

Reliable Analysis of the Interaction between Specific Ligands and Immobilized Beta-2-Adrenoceptor by Adsorption Energy Distribution.

Although a comparatively robust method, immobilized protein-based techniques have displayed limited precision and inconsistent results due to a lack of strategy for the accurate selection of drug adsorption models on the protein surface. We generated the adsorption data of three drugs on immobilized beta-2-adrenoceptor (ß2-AR) by frontal affinity chromatography-mass spectrometry (FAC-MS) and site-specific competitive FAC-MS. Using adsorption energy distribution (AED) calculations, we achieved the best adsorption models for the binding of salbutamol, terbutaline, and pseudoephedrine to immobilized ß2-AR. The Langmuir model proved to be desirable for describing the adsorptions of salbutamol and terbutaline on immobilized ß2-AR, while the bi-Langmuir model was favorable to characterize the adsorption of pseudoephedrine on the receptor. Relying on the accurate determination of association constants, we presented an efficient approach for ß2-AR ligand screening based on the loss of breakthrough time of an indicator drug caused by the inclusion of competitive drugs in the mobile phase. We concluded that the current strategy enables the reliable and accurate analysis of G protein-coupled receptor (GPCR)-drug interaction. The percentage change in the breakthrough time for drugs can provide useful information for estimating their binding affinity to the receptor. This approach builds a powerful platform for high-throughput ligand screening.

One-step methodology for the direct covalent capture of GPCRs from complex matrices onto solid surfaces based on the bioorthogonal reaction between haloalkane dehalogenase and chloroalkanes.

Protein immobilization techniques play an important role in the development of assays for disease diagnosis and drug discovery. However, many of these approaches are not applicable to transmembrane proteins. G protein-coupled receptors (GPCRs) are the largest protein superfamily encoded by the human genome and are targeted by a quarter of all prescription drugs. GPCRs are highly dynamic and sensitive to changes in the ambient environment, and current immobilization methodologies are not suitable for GPCRs. We used haloalkane dehalogenase (Halo) as an immobilization tag fused to the ß2-adrenoceptor (ß2-AR), angiotensin II type 1 (AT1) and angiotensin II type 2 (AT2) receptors. The engineered Halo-tag covalently binds to a specific substrate chloroalkane through Asp 106 in the catalytic pocket. The Halo-tagged GPCRs were expressed in Escherichia coli at a suitable yield. Accordingly, we loaded cell lysate containing Halo-tagged GPCRs onto a macroporous silica gel coated with chloroalkane. Morphological characterization indicated a homogeneous monolayer of immobilized Halo-tagged GPCRs on the silica gel surface. The immobilized receptors proved to be surrounded by specific bound phospholipids including PG C18:1/C18:1. We observed a radio-ligand binding ability and ligand-induced conformational changes in the immobilized GPCRs, suggesting the preservation of bioactivity. This method is a one-step approach for the specific immobilization of GPCRs from cell lysates and validates that immobilized receptors retain canonical ligand binding capacity. Our immobilization strategy circumvents labor-intensive purification procedures and minimizes loss of activity. The immobilized receptors can be applied to high-throughput drug and interaction partner screening for GPCRs.

Bioactive compounds of Shuang-Huang-Lian prescription and an insight into its binding mechanism by ß2 -adrenoceptor chromatography coupled with site-directed molecular docking.

Owing to the promising clinical efficacy and relatively simple composition, Shuang-Huang-Lian prescription is widely prescribed for the treatment of acute upper respiratory tract infection and acute bronchitis in practice. This necessitates the understanding of the bioactive compounds of the prescription and their binding mechanism to ß2 -adrenoceptor, which mediates the aforementioned ailments. In this work, a column containing immobilized ß2 -adrenoceptor was prepared using a diazonium salt reaction. The bioactive compound collected from the ß2 -adrenoceptor column was identified as chlorogenic acid by using high-performance liquid chromatography coupled with ion trap mass spectrometry. Using an injection amount dependent method, chlorogenic acid proved the binding to ß2 -adrenoceptor through two kinds of sites. The numbers of the sites were (1.42 ± 0.03) × 10-8 and (9.06 ± 0.49) × 10-8  M. The association constants were (2.72 ± 0.01) × 105 and (2.80 ± 0.01) × 104  M-1 , respectively. Molecular docking analysis of the interaction between chlorogenic acid and ß2 -adrenoceptor indicated that the binding mainly occurred on Ser169 , Ser173 , and Phe287 of ß2 -adrenoceptor. These results paved the way to screen bioactive compounds of other traditional medicines by receptor chromatography.

Rapid analysis of interaction between six drugs and ß2 -adrenergic receptor by injection amount-dependent method.

Drug-protein interaction analysis has become a considerable topic in life science which includes clarifying protein functions, explaining drug action mechanisms and uncovering novel drug candidates. This work was to determine the association constants (KA ) of six drugs to ß2 -adrenergic receptor by injection amount-dependent method using stationary phase containing the immobilized receptor. The values of KA were calculated to be (25.85 ± 0.035) × 104 m-1 for clorprenaline, (42.51 ± 0.054) × 104 m-1 for clenbuterol, (6.67 ± 0.008) × 104 m-1 for terbutaline, (33.99 ± 0.025) × 104 m-1 for tulobuterol, (7.59 ± 0.011) × 104 m-1 for salbutamol and (78.52 ± 0.087) × 104 m-1 for bambuterol. This rank order agreed well with the data determined by zonal elution, frontal analysis and nonlinear chromatography, even using different batches of ß2 -AR column. A good correlation was found between the association constants by the current method and radio-ligand binding assay. Our data indicates that the injection amount-dependent method is a powerful alternative for rapid analysis of ligand-receptor interactions.

Identifying the antiasthmatic target of doxofylline using immobilized ß2 -adrenoceptor based high-performance affinity chromatography and site-directed molecular docking.

As a xanthine derivative, doxofylline is believed to be dominant for fighting against asthma in practice. Unlike other xanthines, the antiasthmatic effects of doxofylline lack any definite proof of target and mediating mechanism according to previous reports. In this work, the interaction between doxofylline and ß2 -AR was investigated by high performance affinity chromatography using frontal analysis and nonlinear model. The methodology involved the immobilization of ß2 -AR on the silica gel by a random linking method, the determination of the binding parameters by frontal analysis and nonlinear chromatography and the exploration of the binding mechanism by site-directed molecular docking. The association constant for doxofylline binding to immobilized ß2 -AR was determined to be 7.70 × 10(4) M(-1) by nonlinear chromatography and 5.91 × 10(4) M(-1) by frontal analysis. Ser(169) and Ser(173) were the binding sites for the receptor-drug interaction on which hydrogen bond was believed to be the main driven force during the interaction. These results indicated that the antiasthmatic effects of doxofylline may be behind the mediating mechanism of ß2 -AR. High performance affinity chromatography based on immobilized receptor has potential to become an alternative for drug target confirmation and drug-receptor interaction analysis. Copyright © 2016 John Wiley & Sons, Ltd.