The binding of a polymeric ligand to a cell surface receptor can promote its internalization. Methods to track and visualize multivalent ligands within a cell can give rise to new therapeutic strategies and illuminate signaling processes. We have used the features of the ring-opening metathesis polymerization (ROMP) to develop a general strategy for synthesizing multivalent ligands equipped with a latent fluorophore. The utility of ligands of this type is highlighted by visualizing multivalent antigen internalization in live B cells.Advances in organic and polymer chemistry are providing access to new classes of bioactive polymers. 1 With the ability to generate synthetic macromolecules that vary in structure and physical properties, the applications of polymers have expanded rapidly. Some uses include as substrates for cell growth and differentiation, 2 vehicles for drug delivery, 3 therapeutics, 4 and molecules for studying cell signaling. 5 For some biological applications, bioactive polymers must be taken up into cells. Still, little is known about the cellular internalization and trafficking of polymers. Here, we present an approach for following these processes.The bioactive polymers that we focused upon were those generated by ruthenium carbeneinitiated ring-opening metathesis polymerization (ROMP). It was shown that ROMP could be used to generate biologically-active polymers over 10 years ago. 6 Since that time, ROMP has been used produce materials with a variety of biological activities. 7 Continuous advances in catalyst development and polymerization techniques have provided methods to control key features of polymer structure, including their length and the functional groups they present. 8 Ruthenium carbene initiators tolerate a diverse array of functional groups; therefore, they can be used synthesize highly functionalized polymers with tailored biological activities. ROMP initially was used to generate polymers that act on the outside of the cell, but more recently, it has been employed to assemble compounds that can be internalized by cells. Our interest in polymer uptake was prompted by our studies of B cell signaling. 9 For example, polymers generated by ROMP that display antigenic epitopes can promote antibody production in vivo. The ability of these polymers to activate this process depends upon their interactions with the antigen-specific B cell receptor (BCR), a membrane-bound immunoglobulin on the surface of B cells. These polymers bind to the specific B cell receptor to activate signaling but they also promote its internalization. We therefore anticipated that, like other antigens, these polymers would be taken up by endocytosis. To investigate polymer internalization, we envisioned using antigenic polymers equipped with a group that would report directly on internalization.Most approaches to following ligand internalization rely on appending a fluorescent dye to the molecule of interest. Uptake is detected using a discontinuous assay (typically fluorescence microscopy). Becaus...
Three indicator displacement assays are described for the detection of phosphatidylserine in a bilayer membrane. A series of Zn 2+ -dipicolylamine coordination compounds are used to bind selectively to the phosphatidylserine and act as a colorimetric chemosensing ensemble when combined with the UV-Vis indictor pyrocatechol violet. A similar displacement assay uses a coumarin methylsulfonate derivative as a fluorescent indicator, and a third assay involves quenching of calcein fluorescence by Cu 2+ and subsequent fluorescence restoration upon addition of phosphatidylserine. In the best case, vesicle membranes containing as little as 5% phosphatidylserine could be detected under physiologically relevant conditions using as little as 10 mM sensing ensemble, and two of the three systems allow vesicles containing 50% phosphatidylserine to be detected by the naked eye.
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