Synthesis and Application of Fluorescein-Labeled Pluronic Block Copolymers to the Study of Polymer−Surface Interactions

Abstract

We present a novel methodology for the conjugation of Pluronic poly(ethylene oxide)−poly(propylene oxide)−poly(ethylene oxide) block copolymers with a fluorescein derivative, 5-(4,6-dichlorotriazinyl) aminofluorescein (5-DTAF), at room temperature under aqueous conditions. Two Pluronic block copolymers, F68 with molecular weight (MW) 8400 and P105 with MW 6500, were examined. The labeled block copolymers exhibited maximum absorbance at 498 nm, peak fluorescence at 516 nm, and a marked reduction in both absorbance and fluorescence at acidic pH below 7. Flow cytometry was employed to quantify the real-time adsorption kinetics of labeled block copolymers to two types of surfaces: polystyrene microspheres and blood cells, specifically platelets. This methodology allowed detection of Pluronics at low solution concentrations down to 1 μM. In the case of labeled Pluronic F68 and P105 binding to polystyrene beads, a rapid time-dependent adsorption kinetics was observed with 80% adsorption occurring within 30 s and saturation in 2 min. The concentration-dependent adsorption profile for Pluronic F68 binding to polystyrene beads was similar to that of P105, with increasing binding in the range from 1 to 25 μM and saturation at higher concentrations. Surface modification of the polystyrene beads by carboxylation dramatically reduced the binding of both Pluronic F68 and P105. In experiments that examined Pluronic interaction with blood platelets, we observed that Pluronics not only bind the platelets in a dose-dependent fashion but also bind soluble macromolecules in blood plasma. Overall, the labeled block copolymers synthesized in this study were found to exhibit the fluorescence characteristics of the unconjugated 5-DTAF and the binding characteristics of unlabeled Pluronics. Such an approach of labeling hydroxyl-terminated polymers with fluorescein and applying flow cytometry could be used to probe polymer−surface interactions in a variety of chemical and biological systems.