No abstract

Keratinocyte growth factor 1 (KGF-1) has proven useful in the treatment of pathologies associated with dermal adnexae, liver, lung, and the gastrointestinal tract diseases. However, poor stability and short plasma half-life of the protein have restricted its therapeutic applications. While it is possible to improve the stability and extend the circulating half-life of recombinant human KGF-1 (rhKGF-1) using solution-phase PEGylation, such preparations have heterogeneous structures and often low specific activities due to multiple and/or uncontrolled PEGylation. In the present study, a novel solid-phase PEGylation strategy was employed to produce homogenous mono-PEGylated rhKGF-1. RhKGF-1 protein was immobilized on a Heparin-Sepharose column and then a site-selective PEGylation reaction was carried out by a reductive alkylation at the N-terminal amino acid of the protein. The mono-PEGylated rhKGF-1, which accounted for over 40% of the total rhKGF-1 used in the PEGylation reaction, was purified to homogeneity by SP Sepharose ion-exchange chromatography. Our biophysical and biochemical studies demonstrated that the solid-phase PEGylation significantly enhanced the in vitro and in vivo biostability without affecting the over all structure of the protein. Furthermore, pharmacokinetic analysis showed that modified rhKGF-1 had considerably longer plasma half-life than its intact counterpart. Our cell-based analysis showed that, similar to rhKGF-1, PEGylated rhKGF-1 induced proliferation in NIH 3T3 cells through the activation of MAPK/Erk pathway. Notably, PEGylated rhKGF-1 exhibited a greater hepatoprotection against CCl4-induced injury in rats compared to rhKGF-1.

Diethylstilbestrol (DES), an endocrine disrupting chemical, has been linked to serious health problems in humans. In this work, a regenerative flexible upconversion-fluorescence biosensor was designed for the detection of DES in foodstuffs and environmental samples. Herein, amino-functionalized upconversion nanoparticles (UCNPs) were synthesized and immobilized on the surface of a flexible polydimethylsiloxane substrate, which was further modified with complementary DNA and dabcyl-labeled DES aptamer. The fluorescence resonance energy transfer (FRET) system was established for DES detection between dabcyl and UCNPs as the acceptor and donor pairs, respectively, which resulted in the quenching of the upconversion luminescence intensity. In the presence of a target, the FRET system was destroyed and upconversion fluorescence was restored due to the stronger affinity of the aptamer toward DES. The designed biosensor was also implemented in a dual-mode signal readout based on images from a smartphone and spectra from a spectrometer. Under the optimized experimental conditions, good linear relationships were achieved based on imaging (y = 53.055x + 36.175, R 2 = 0.9851) and spectral data (y = 1.1582x + 1.9561, R 2 = 0.9897). The designed biosensor revealed great practicability with a spiked recovery rate of 77.91–97.95% for DES detection in real environment and foodstuff samples. Furthermore, the proposed biosensor was regenerated seven times with an accuracy threshold of 80% demonstrating its durability and reusability. Thus, this biosensor is expected to be applied to point-of-care and on-site detection based on the developed portable smartphone device and android application.

Background Gefitinib is an orally potent and selective ATP-competitive inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase and is commonly used to treat locally advanced or metastatic non-small-cell lung cancer (NSCLC) with sensitive EGFR mutations. Multiple adverse effects associated with gefitinib, including liver and lung injuries, severe nausea, and diarrhea, have limited its clinical application. Xenobiotic-induced bioactivation is thought to be an important reason for gefitinib toxicity, which encouraged us to clarify the metabolism of gefitinib in NSCLC patients. Materials and methods An ultra-performance liquid chromatography coupled with triple quadrupole time-of-flight mass spectrometry (UPLCQ-TOF-MS) method was established to tentatively identify the metabolites of gefitinib in human plasma. The extracted ion chromatogram peak intensity threshold was set at 1500 cps with minimum MS and MS/MS peak intensities of 400 and 100 cps, respectively. Results A total of 18 tentative metabolites were identified. Eight novel tentative metabolites with metabolic changes in dechlorination, defluorination, and hydrogenation on the quinazoline skeleton; removal of a partial or complete 3-chloro-4-fluoroaniline-substituted group; and sulfate