Evaluation of phototoxicity of dendritic porphyrin-based phosphorescent oxygen probes: an in vitro study

Abstract

Biological oxygen measurements by phosphorescence quenching make use of exogenous phosphorescent probes, which are introduced directly into the medium of interest (e.g. blood or interstitial fluid) where they serve as molecular sensors for oxygen. The byproduct of the quenching reaction is singlet oxygen, a highly reactive species capable of damaging biological tissue. Consequently, potential probe phototoxicity is a concern for biological applications. Herein, we compared the ability of polyethyleneglycol (PEG)-coated Pd tetrabenzoporphyrin (PdTBP)-based dendritic nanoprobes of three successive generations to sensitize singlet oxygen. It was found that the size of the dendrimer has practically no effect on the singlet oxygen sensitization efficiency in spite of the strong attenuation of the triplet quenching rate with an increase in the dendrimer generation. This unexpected result is due to the fact that the lifetime of the PdTBP triplet state in the absence of oxygen increases with dendritic generation, thus compensating for the concomitant decrease in the rate of quenching. Nevertheless, in spite of their ability to sensitize singlet oxygen, the phosphorescent probes were found to be non-phototoxic when compared with the commonly used photodynamic drug Photofrin in a standard cell-survival assay. The lack of phototoxicity is presumably due to the inability of PEGylated probes to associate with cell surfaces and/or penetrate cellular membranes. In contrast, conventional photosensitizers bind to cell components and act by generating singlet oxygen inside or in the immediate vicinity of cellular organelles. Therefore, PEGylated dendritic probes are safe to use for tissue oxygen measurements as long as the light doses are less than or equal to those commonly employed in photodynamic therapy.

Fig. 1

Structures of phosphorescent PdTBP-based dendrimers. PEG designates residues of polyethyleneglycol monomethyl ether (av. MW 350).

Fig. 2

Absorption spectra of compounds G0–G3 in buffered aqueous solutions at 298 K.

Fig. 3

Phosphorescence spectra in air-equilibrated aqueous solutions at 298 K. The emission intensities are directly comparable since the spectra were recorded for isoabsorbing solutions at the excitation wavelength (λ_ex = 634 nm).

Fig. 4

Stern–Volmer oxygen phosphorescence quenching plots for compounds G0–G3 in aqueous solutions at 295 K. Lines show the fits of the raw data to the linear equations. Phosphorescence decays were analyzed by two-exponential functions; apparent lifetimes τ were obtained by intensity-weighted averaging.

Fig. 5

Phosphorescence of singlet oxygen in air-equilibrated C₂H₅OH–CH₃OH 4 : 1 (v/v) solutions (λ_max = 1270 nm) as sensitized by compounds G0–G3. The tail phosphorescence of Pd porphyrin-dendrimers was subtracted from the spectra (see Fig. S1, S2 (ESI) for the raw data). The emission intensities are directly comparable, since the absorbances of the sensitizer solutions at the excitation wavelength (635 nm) were kept equal for all the compounds.

Fig. 6

Clonogenic survival data for RIF cells incubated with Photofrin or G2 during 18 h and irradiated with 630 nm light. Hollow symbols – photosensitizer is absent from the medium during irradiation. Filled symbols – photosensitizer is left in the medium during irradiation. Surviving fractions are calculated as fractions of the colony-forming cells in the treated samples compared to controls that received neither photosensitizer nor light. The data are shown as average ± SE from 3 independent studies. * No surviving cells were found for this condition – the point indicates the lower limit of the assay sensitivity.

Scheme 1

Diagram of photophysical processes occurring in phosphorescent molecules: hν – excitation, isc – intersystem crossing; k₀ – rate constant of phosphorescence emission (k₀ = 1/τ₀ = k_r + k_nr, where k_r and k_nr are the rate constants of the radiate and non-radiative deactivation of the triplet state in the absence of oxygen); k_q – rate constant of bimolecular quenching, which results in conversion of ground state oxygen O₂(X³Σ_g^-) into excited singlet state oxygen O₂(a¹Δ_g).