Synthesis and application of fluorescein-labeled Pluronic block copolymersto the study of polymer-surface interactions

We present a novel methodology for the conjugation of Pluronic poly(ethylen e 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 copolym ers, F68 with molecular weight (MW) 8400 and P105 with MW 6500, were examin ed. The labeled block copolymers exhibited maximum absorbance at 498 nm, pe ak fluorescence at 516 nm, and a marked reduction in both absorbance and fl uorescence at acidic pH below 7. Flow cytometry was employed to quantify th e 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 concentra tions down to 1 muM. In the case of labeled Pluronic F68 and P105 binding t o polystyrene beads, a rapid time-dependent adsorption kinetics was observe d with 80% adsorption occurring within 30 s and saturation in 2 min. The co ncentration-dependent adsorption profile for Pluronic F68 binding to polyst yrene beads was similar to that of P105, with increasing binding in the ran ge from 1 to 25 muM and saturation at higher concentrations. Surface modifi cation of the polystyrene beads by carboxylation dramatically reduced the b inding 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 macromolec ules in blood plasma. Overall, the labeled block copolymers synthesized in this study were found to exhibit the fluorescence characteristics of the un conjugated 5-DTAF and the binding characteristics of unlabeled Pluronics. S uch an approach of labeling hydroxyl-terminated polymers with fluorescein a nd applying flow cytometry could be used to probe polymer-surface interacti ons in a variety of chemical and biological systems.