Epidermal growth factor receptor (EGFR) is often constitutively stimulated in many cancers owing to the binding of ligands such as epidermal growth factor (EGF). Therefore, it is necessary to investigate the interaction between EGFR and its targeting biomolecules. The main aim of this study was to estimate the binding affinity and adhesion force of two targeting molecules, anti-EGFR monoclonal antibody (mAb LA1) and the peptide GE11 (YHWYGYTPQNVI), with respect to EGFR and to compare these values with those obtained for the ligand, EGF. Surface plasmon resonance (SPR) was used to determine the equilibrium dissociation constant (KD) for evaluating the binding affinity. Atomic force microscopy (AFM) was performed to estimate the adhesion force. In the case of EGFR, the KD of EGF, GE11, and mAb LA1 were 1.77 × 10−7, 4.59 × 10−4 and 2.07 × 10−9, respectively, indicating that the binding affinity of mAb LA1 to EGFR was higher than that of EGF, while the binding affinity of GE11 to EGFR was the lowest among the three molecules. The adhesion force between EGFR and mAb LA1 was 210.99 pN, which is higher than that observed for EGF (209.41 pN), while the adhesion force between GE11 and EGFR was the lowest (59.51 pN). These results suggest that mAb LA1 binds to EGFR with higher binding affinity than EGF and GE11. Moreover, the adhesion force between mAb LA1 and EGFR was greater than that observed for EGF and GE11. The SPR and AFM experiments confirmed the interaction between the receptor and targeting molecules. The results of this study might aid the screening of ligands for receptor targeting and drug delivery.

BackgroundDuring the onset of osteoarthritis (OA), certain biochemical events have been shown to accelerate cartilage degradation, including the dysregulation of cartilage ECM anabolism, abnormal generation of reactive oxygen species (ROS) and overproduction of proteolytic enzymes and inflammatory cytokines. The potency of aucubin in protecting cellular components against oxidative stress, inflammation and apoptosis effects are well documented, which makes it a potential candidate for OA treatment. In this study, we aimed to evaluate the protective benefits of aucubin against OA using H2O2 and compression induced OA-like chondrocyte models.MethodsThe effects of aucubin were studied in porcine chondrocytes after 1 mM H2O2 stimulation for 30 min or sustained compression for 24 h. Effects of aucubin on cell proliferation and cytotoxicity of chondrocytes were measured with WST-1 and LDH assays. ROS production was evaluated by the Total ROS/Superoxide Detection Kit. Caspase-3 activity was evaluated by the CaspACE assay system. The levels of apoptosis were evaluated by the Annexin V-FITC apoptosis detection kit. OA-related gene expression was measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Total DNA quantification was evaluated by the DNeasy Blood and Tissue kit. Sulfated-glycosaminoglycans (sGAGs) production and content were evaluated by DMMB assay and Alcian blue staining.ResultsThe results showed that the ROS scavenge effects of aucubin appeared after 1 h of pretreatment. Aucubin could reduce the caspase-3 activity induced by H2O2, and reduced the apoptosis cell population in flowcytometry. In RT-qPCR results, aucubin could maintain ACAN and COL2A1 gene expressions, and prevent IL6 and MMP13 gene up-regulation induced by H2O2 and compression stimulations. In the DMMB assay and Alcian blue staining, aucubin could maintain the sGAG content and protect chondrocytes against compressive stress, but not oxidative stress from H2O2.ConclusionsThese results indicated that aucubin has protective effects in an osteoarthritic chondrocyte model induced by H2O2 and mechanical stimulus.

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The lack of an effective treatment for osteoarthritis (OA) reflects the great need for alternative therapies and drug discovery. Disease models can be used for earlystage compound screening and disease studies. Chondrocytes are solely responsible for the maintenance of the articular cartilage extracellular matrix. Our strategy involved the generation of a cell-based model of OA, a more readily studied disease. Instead of using animal cartilage explants, we incorporated isolated porcine chondrocytes with hydrogel to form threedimensional assemblies. We could identify the specific magnitude-dependent metabolic responses of chondrocytes by applying a series of static and cyclic compression, and therefore successfully generated a novel OA-like cell-based model for drug screening. AbstractMechanical stress damage and insufficient self-repair can contribute to osteoarthritis (OA) in the affected joint. As the effects of stress on chondrocyte metabolism can regulate cartilage homeostasis, the specific stress-response condition is therefore a key to the generation of an OA disease model. We aimed to produce a specific stress-and cell-based OA model after evaluating the metabolic responses of chondrocytes in response to a series of static and cyclic compression stressors. A static load exceeding 40 psi initiated extracellular matrix (ECM) degradation through a decrease in the sulphated-glycosaminoglycan (GAG) content, upregulation of catabolic matrix metalloproteinase (MMP)-13 encoding gene expression, and downregulation of the ECM-related aggrecan and type II collagen encoding genes within 24 h. Indicators of pro-inflammatory events and oxidative stress were found to correlate with increased IL-6 expression and reactive oxygen species (ROS) production, respectively. However, chondrocytes stimulated by moderate cyclic loading (30-40 psi) exhibited increased ECM-related gene expression without significant changes in catabolic and pro-inflammatory gene expression. BMP-7 expression increased at cyclic loading levels above 30-60 psi. These results demonstrated that static compression exceeding 60 psi is sufficient to produce OA-like chondrocytes that exhibit signs of ECM degradation and inflammation. These OA-like chondrocytes could therefore be used as a novel cell-based drug screening system.

Cancer is the leading cause of mortality worldwide, and lung cancer is the most malignant. However, the high failure rate in oncology drug development from in vitro studies to in vivo preclinical models indicates that the modern methods of evaluating drug efficacies in vitro are not reliable. Traditional 2D cell culture has proved inadequate to mimic real physiological conditions. Current 3D cell culture methods do not represent the delicate structure of lung alveoli. To mimic lung alveoli structure, a cell-containing enzyme-crosslinked gelatin microbubble scaffold was produced by mixing surfactant-containing gelatin solution with microbial transglutaminase (mTGase)-mixed A549 cell suspension in a four-channel flow-focusing microfluidic device. With uniform pore size of about 100 μm in diameter, this gelatin microbubble scaffold resembled the lung alveoli in structure and in mechanical properties with good biocompatibility. Effective gemcitabine concentration required to induce cell death in microbubble scaffolds was significantly higher than in 2D culture together with a longer treatment time. Cell death mechanisms were confirmed to be gemcitabine-induced cell apoptosis through Western blotting and real-time polymerase chain reaction. H&E staining and TUNEL assay showed rounded cells with DNA damage in drug-treated scaffolds. Taken together, the cell-containing microbubble scaffolds successfully mimicked lung alveoli in structure and cellular responses after gemcitabine treatment were similar to clinical regimen of treating lung carcinoma. The microbubble scaffold is promising to facilitate anticancer drug discovery by providing more accurate preclinical predictions.