Fluorescence properties of doxorubicin in PBS buffer and PVA films

Abstract

We studied steady-state and time-resolved fluorescence properties of an anticancer drug Doxorubicin in a saline buffer and poly-vinyl alcohol (PVA) film. Absorption of Doxorubicin, located at blue-green spectral region, allows a convenient excitation with visible light emitting diodes or laser diodes. Emission of Doxorubicin with maximum near 600nm can be easily detected with photomultipliers and CCD cameras. Both, absorption and fluorescence spectra in polymeric matrix show more pronounced vibronic structures than in solution. Also, the steady-state anisotropy in the polymer film is significantly higher than in the saline solution. In PVA film the fluorescence anisotropy is about 0.30 whereas in the saline buffer only 0.07. Quantum efficiencies of Doxorubicin were compared to a known standard Rhodamine 101 which has fluorescence emission in a similar spectral region. The quantum yield of Doxorubicin in PVA film is more than 10% and about twice higher than in the saline solution. Similarly, the lifetime of doxorubicin in PVA film is about 2ns whereas in the saline solution only about 1ns. The fluorescence anisotropy decays show that Doxorubicin molecules are freely rotating in the saline buffer with a correlation time of about 290ps, and are almost completely immobilized in the PVA film. The spectroscopic investigations presented in this manuscript are important, as they provide answers to changes in molecular properties of Doxorubicin depending changes in the local environment, which is useful when synthesizing nanoparticles for Doxorubicin entrapment.

Fig. 1

Absorption spectra of Doxorubicin in PBS buffer (left) and PVA film (right).

Fig. 2

Excitation and fluorescence spectra of Doxorubicin in PBS buffer (left) and PVA film (right). Also shown are excitation and emission anisotropies. The fluorescence excitation spectra were measured at 600 nm observation and fluorescence emission spectra were excited at 500 nm.

Fig. 3

Comparison of Doxorubicin and Rhodamine 101 emission spectra at 500 nm excitation. Left: in PBS buffer; Right: in PVA films. The spectra were corrected for the amount of absorbed light, see Eq. (2).

Fig. 4

Fluorescence intensity decays of Doxorubicin in PBS buffer (left) and PVA film (right). In the case of PVA film Doxorubicin fluorescence intensity decay requires two exponents in order to fit the data.

Fig. 5

Fluorescence anisotropy decays of Doxorubicin in PBS buffer and PVA film. The measurements were done at room temperature, using a 470 nm laser diode for excitation.

Fig. 6

Fluorescence detectability limit of Doxorubicin in PBS buffer. Measurements were performed in 1 cm × 1 cm cuvette at room temperature. The samples were excited at 470 nm.

Scheme 1

Chemical structure of Doxorubicin.

Scheme 2

Front face geometry set-up for steady-state measurements of Doxorubicin trapped in PVA film.