Biophysics Department, Faculty of Science, Cairo University, Giza, Egypt
Abstract. Fluorescein diphosphate (FDP) is a fluorogenic alkaline phosphatase substrate that has recently been isolated in pure form and made commercially available. FDP is colorless and nonfluorescent. Sequential alkaline phosphatase-mediated hydrolysis of the two phosphate substituents yields weakly fluorescent fluorescein monophosphate followed by strongly fluorescent fluorescein (excitation/emission ~ 490/514 nm). The long-term motivation behind this work was to develop methods in which chemical reactions can be initiated and followed in complex liposomes synthesized using combinatorial schemes so that rates and mechanisms of chemical reactions can be studied in environments that approximate true cellular environments. In this work negatively charged fluorescein diphosphate was encapsulated in thermosensitive liposomes composed of Dipalmitoylphosphatidylcholine (DPPC), Hydrogenated Soy phosphatidylcholine (HSPC), Cholesterol (Chol) at a molar ratio of (1:1:1). The release of FDP in response to 42 ºC heating was followed in a medium containing 10% serum and alkaline phosphatase enzyme. More that 60% of the encapsulated FDP was released in the outer medium in response to heating, once released, It emits strong fluorescent light at a wavelength of 514 nm indicating complete hydrolysis to FDP, yielding fluorescein monophosphate which emits fluorescent light upon excitation at a wavelength of 490 nm. The phase transition temperature of the liposomes was followed using a differentiated fluorescence scan. Right angle light scattering intensity at 436 nm per unit lipid concentration was measured. The optical parameters were explained by the Rayleigh – Gans – Debye theory in which the liposomes were modeled as homogenous spheres with mean refractive indices determined by the volume fractions of the lipids in vesicles.
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