Extracellular vesicles (EVs), including exosomes and microvesicles (<200 nm), play a vital role in intercellular communication and carry a net negative surface charge under physiological conditions. Zeta potential (ZP) is a popular method to measure the surface potential of EVs, while used as an indicator of surface charge, and colloidal stability influenced by surface chemistry, bioconjugation, and the theoretical model applied. Here, we investigated the effects of such factors on ZP of well-characterized EVs derived from the human choriocarcinoma JAr cells. The EVs were suspended in phosphate-buffered saline (PBS) of various phosphate ionic concentrations (0.01, 0.1, and 1 mM), with or without detergent (Tween-20), or in the presence (10 mM) of different salts (NaCl, KCl, CaCl 2 , and AlCl 3 ) and at different pH values (4, 7, and 10) while the ZP was measured. The ZP changed inversely with the buffer concentration, while Tween-20 caused a significant ( p < 0.05) lowering of the ZP. Moreover, the ZP was significantly ( p < 0.05) less negative in the presence of ions with higher valency (Al 3+ /Ca 2+ ) than in the presence of monovalent ones (Na + /K + ). Besides, the ZP of EVs became less negative at acidic pH, and vice versa . The integrated data underpins the crucial role of physicochemical attributes that influence the colloidal stability of EVs.

Tumor-associated macrophages (TAMs) expressing the multi-ligand endocytic receptor mannose receptor (CD206/MRC1) contribute to tumor immunosuppression, angiogenesis, metastasis, and relapse. Here, we describe a peptide that selectively targets MRC1-expressing TAMs (MEMs). We performed in vivo peptide phage display screens in mice bearing 4T1 metastatic breast tumors to identify peptides that target peritoneal macrophages. Deep sequencing of the peptide-encoding inserts in the selected phage pool revealed enrichment of the peptide CSPGAKVRC (codenamed “UNO”). Intravenously injected FAM-labeled UNO (FAM-UNO) homed to tumor and sentinel lymph node MEMs in different cancer models: 4T1 and MCF-7 breast carcinoma, B16F10 melanoma, WT-GBM glioma and MKN45-P gastric carcinoma. Fluorescence anisotropy assay showed that FAM-UNO interacts with recombinant CD206 when subjected to reducing conditions. Interestingly, the GSPGAK motif is present in all CD206-binding collagens. FAM-UNO was able to transport drug-loaded nanoparticles into MEMs, whereas particles without the peptide were not taken up by MEMs. In ex vivo organ imaging, FAM-UNO showed significantly higher accumulation in sentinel lymph nodes than a control peptide. This study suggests applications for UNO peptide in diagnostic imaging and therapeutic targeting of MEMs in solid tumors.

Over the past decade the kinetics of ligand binding to a receptor have received increasing interest. The concept of drug-target residence time is becoming an invaluable parameter for drug optimization. It holds great promise for drug development, and its optimization is thought to reduce off-target effects. The success of long-acting drugs like tiotropium support this hypothesis. Nonetheless, we know surprisingly little about the dynamics and the molecular detail of the drug binding process. Because protein dynamics and adaptation during the binding event will change the conformation of the protein, ligand binding will not be the static process that is often described. This can cause problems because simple mathematical models often fail to adequately describe the dynamics of the binding process. In this minireview we will discuss the current situation with an emphasis on G-protein-coupled receptors. These are important membrane protein drug targets that undergo conformational changes upon agonist binding to communicate signaling information across the plasma membrane of cells.

We present here the implementation of budded baculoviruses that display G protein-coupled receptors on their surfaces for the investigation of ligand-receptor interactions using fluorescence anisotropy (FA). Melanocortin 4 (MC4) receptors and the fluorescent ligand Cy3B-NDP-α-MSH were used as the model system. The real-time monitoring of reactions and the high assay quality allow the application of global data analysis with kinetic mechanistic models that take into account the effect of nonspecific interactions and the depletion of the fluorescent ligand during the reaction. The receptor concentration, affinity and kinetic parameters of fluorescent ligand binding as well as state anisotropies for different fluorescent ligand populations were determined. At low Cy3B-NDP-α-MSH concentrations, a one-site receptor-ligand binding model described the processes, whereas divergence from this model was observed at higher ligand concentrations, which indicated a more complex mechanism of interactions similar to those mechanisms that have been found in experiments with radioactive ligands. The information obtained from our kinetic experiments and the inherent flexibility of FA assays also allowed the estimation of binding parameters for several MC4 receptor-specific unlabelled compounds. In summary, the FA assay that was developed with budded baculoviruses led the experimental data to a level that would solve complex models of receptor-ligand interactions also for other receptor systems and would become as a valuable tool for the screening of pharmacologically active compounds.

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