Time-resolved fluorescence and fluorescence anisotropy of fluorescein-labeled poly(N-isopropylacrylamide) incorporated in polymersomes

The phase behavior of fluorescein isothiocyanate (FITC) labeled poly(N-isopropylacrylamide) (PNIPAAm) incorporated in polymersomes (Ps) was studied by monitoring the fluorescence lifetime (FL) and the time-resolved fluorescence anisotropy (TRFA) as a function of temperature at pH 7.4. Ps containing FITC-labeled PNIPAAm with a diameter less than 200 nm were prepared by injecting a THF solution of poly(ethylene glycol)-b-poly(d,l-lactide) (mPEG-PDLLA) and FITC tagged PNIPAAm (FITC-N) into phosphate buffered saline (PBS, pH 7.4). Solutions of free FITC (2 µM) and FITC-N (2 µM) in PBS were used as controls. The polarized fluorescence decay curves of FITC were fitted with one rotational correlation time (¿1) and the corresponding amplitude (ß1), while those for FITC-N were fitted with two rotational correlation times (¿1,2) and their corresponding amplitudes (ß1,2). Short rotational correlation times, ¿1, correspond with the rotation of the FITC molecule itself, whereas ¿2 corresponds to FITC-segmental rotation. FITC-N encapsulated in Ps (FITC-N/Ps) showed a decrease of the rotational motion upon increasing the temperature. The long rotational correlation time (¿2) of FITC-N increased 3 fold, going from 15 to 40 °C, reflecting a reduced rotational mobility. The residual anisotropy (ß8) of FITC-N/Ps at pH 7.4 showed a gradual increase, going from 15 to 25 °C followed by a gradual decrease at higher temperatures. These results are explained by a transition from coil to globule, a gradual increase of intermolecular aggregation, and possibly phase separation and hydrogel formation