Added Value of Scintillating Element in Cerenkov-Induced Photodynamic Therapy

Abstract

Cerenkov-induced photodynamic therapy (CR-PDT) with the use of Gallium-68 (⁶⁸Ga) as an unsealed radioactive source has been proposed as an alternative strategy to X-ray-induced photodynamic therapy (X-PDT). This new strategy still aims to produce a photodynamic effect with the use of nanoparticles, namely, AGuIX. Recently, we replaced Gd from the AGuIX@ platform with Terbium (Tb) as a nanoscintillator and added 5-(4-carboxyphenyl succinimide ester)-10,15,20-triphenylporphyrin (P1) as a photosensitizer (referred to as AGuIX@Tb-P1). Although Cerenkov luminescence from ⁶⁸Ga positrons is involved in nanoscintillator and photosensitizer activation, the cytotoxic effect obtained by PDT remains controversial. Herein, we tested whether free ⁶⁸Ga could substitute X-rays of X-PDT to obtain a cytotoxic phototherapeutic effect. Results were compared with those obtained with AGuIX@Gd-P1 nanoparticles. We showed, by Monte Carlo simulations, the contribution of Tb scintillation in P1 activation by an energy transfer between Tb and P1 after Cerenkov radiation, compared to the Gd-based nanoparticles. We confirmed the involvement of the type II PDT reaction during ⁶⁸Ga-mediated Cerenkov luminescence, id est, the transfer of photon to AGuIX@Tb-P1 which, in turn, generated P1-mediated singlet oxygen. The effect of ⁶⁸Ga on cell survival was studied by clonogenic assays using human glioblastoma U-251 MG cells. Exposure of pre-treated cells with AGuIX@Tb-P1 to ⁶⁸Ga resulted in the decrease in cell clone formation, unlike AGuIX@Gd-P1. We conclude that CR-PDT could be an alternative of X-PDT.

Keywords: AGuIX; Cerenkov radiation; Gadolinium; Monte Carlo simulations; Terbium; glioblastoma; photodynamic therapy; singlet oxygen.

Scheme 1

Cerenkov radiation-induced photodynamic therapy using Gallium-68. Radioactive decay of Gallium-68 (⁶⁸Ga) isotope results of β⁺ (positron) emission. The particle travels faster than the phase velocity of light in the medium, resulting from Cerenkov luminescence from ultraviolet (UV) to infra-red spectra. Visible Cerenkov photons (yellow) will excite the photosensitizer (PS) directly, whereas UV photons (purple) will be converted by nanoscintillator fluorescence to the visible domain. In addition, at the end of the positron path, positron/electron annihilation produces coincident 511 keV γ-rays. These photons can be absorbed by the nanoscintillator by photoelectric effect to produce scintillation photons, which will be absorbed by the PS, and, finally, the PS could generate singlet oxygen (¹O₂).

Figure 1

Kinetics of singlet oxygen production by AGuIX@Tb-P1 under Gallium-68 exposure. Experiments were performed with 7.35 µM AGuIX@Tb-P1 (P1 equivalent), 10 µM fluorescent probe and 15–20 MBq Gallium-68 (⁶⁸Ga). Probe fluorescence signals were revealed every 5 min during radionuclide irradiation. The production of singlet oxygen was inhibited by adding sodium azide (NaN₃) or bovine serum albumin (BSA) to the reaction mixture. The radioactive decay was estimated and plotted as a function of experiment delay. Three independent experiments were performed; each point corresponds to the mean ± SD.

Figure 2

Slope comparison of singlet oxygen production dynamic and Gallium-68 decay. The Gallium-68 (⁶⁸Ga) decay presented here was determined numerically by applying the exponential decay law whereas singlet oxygen production was assessed by SOSG fluorescence measurements.

Figure 3

Cerenkov luminescence as simulated in Gate. (a) Snapshot of the defined geometry. Cerenkov photons (green) are emitted all along positron paths (blue), which may interact with cells (red) into which a cluster of nanoparticles have been defined (yellow). (b) Cerenkov spectrum (from 250 up to 800 nm) in water inside the main simulation volume.

Figure 4

Luminescence spectra of AGuIX nanoplatform with Terbium (a) and Gadolinium (b) associated with porphyrin. Terbium (Tb) luminescence during Gallium-68 (⁶⁸Ga) irradiation exhibits the 4 characteristic peaks. In presence of porphyrin (P1), these peaks’ intensities decrease, demonstrating the energy transfer from Tb to P1. Simulations were performed on GEANT4-Gate v. 9.1.

Figure 5

Impact of Gallium-68 deposition into U-251 MG cells. Anchorage-dependent clonogenic capability was assayed after cell exposure to increase concentrations of Gallium-68 (⁶⁸Ga) (a). Similarly, cells were pre-treated either in the presence of AGuIX@Tb-P1 or AGuIX@Tb (b) or in the presence of AGuIX@Gd or AGuIX@Gd-P1 (c), for 24 h, before exposure to ⁶⁸Ga for another 24 h delay. Cells were allowed to grow for 7 days. Cell clones obtained were counted after formol fixation and crystal violet staining. Clonogenic capabilities are expressed relative to control cells (b,c). In (c), cells pre-treated with AGuIX@Tb-P1 and irradiated at 2.0 Gy were used as a positive control of PDT efficiency on U-251 MG cell survival (grey box). Results are means ± S.D. (n = 12 wells/condition); significant difference, if any, was evaluated by Kruskal–Wallis test and post hoc by the Dunn test (*** p < 0.001, ** p < 0.01, * p < 0.05).