Organic phosphorescent nanoscintillator for low-dose X-ray-induced photodynamic therapy

X-ray-induced photodynamic therapy utilizes penetrating X-rays to activate reactive oxygen species in deep tissues for cancer treatment, which combines the advantages of photodynamic therapy and radiotherapy. Conventional therapy usually requires heavy-metal-containing inorganic scintillators and organic photosensitizers to generate singlet oxygen. Here, we report a more convenient strategy for X-ray-induced photodynamic therapy based on a class of organic phosphorescence nanoscintillators, that act in a dual capacity as scintillators and photosensitizers. The resulting low dose of 0.4 Gy and negligible adverse effects demonstrate the great potential for the treatment of deep tumours. These findings provide an optional route that leverages the optical properties of purely organic scintillators for deep-tissue photodynamic therapy. Furthermore, these organic nanoscintillators offer an opportunity to expand applications in the fields of biomaterials and nanobiotechnology.

Measurements. 1 H and 13 C nuclear magnetic resonance spectra were collected using a Bruker Ultra Shield Plus spectrometer (400 MHz). Chemical shifts were calibrated by using tetramethylsilane (TMS) in deuterated solvents as the internal standard.
Elemental analysis was accomplished on a Vario EL Cube. Steady-state luminescence and excitation spectra were recorded using Hitachi F-7100 and Edinburgh FLS1000 fluorescence spectrophotometers. The lifetime was obtained on a fluorescence spectrophotometer (Edinburgh FLS1000) equipped with a xenon arc lamp (Xe900), a nanosecond hydrogen flash (nF920), or a microsecond flash (μF900). TEM image was taken on 2100Plus transmission electron microscope (JEOL Ltd., Tokyo, Japan).
Zeta potential was measured on a NanoPlus-3 Particle Analyzer (OTSUKA). The fluorescence images of cells were taken on a laser scanning confocal microscopy (Olympus FV1200, Japan).
Synthesis of 9,9'-(6-(2-iodophenoxy)-1,3,5-triazine-2,4-diyl)bis(9H-carbazole) (ITC). This molecule was prepared according to the previously reported literature S31 . In a nitrogen-filled two-necked flask with 9H-carbazole (5 g, 29.9 mmol), 40 mL dry and degassed THF was slowly injected. Afterward, n-butyllithium (20.5 mL, 1.6 mol/L in hexane) was added dropwise at 273 K. The resulting mixture was stirred at room temperature for 2 hours. Following the same procedure, a solution of 2,4,6trichloro-1,3,5-triazine (2.7 g, 14.9 mmol) in THF (10 mL) was prepared. Subsequently, these two solutions were slowly blended under nitrogen atmosphere at 353 K and stirred for about 8 hours. Once the reaction was completed, the mixture was filtered and washed with cold acetone. Faint yellow powders (9,9'-(6-chloro-1,3,5-triazine-2,4-diyl)bis(9H-carbazole), CzDClT, 3 g, 6.7 mmol) were obtained in 45% yield. To a solution of o-iodophenol (2.2 g, 10.1 mmol) and dry THF (15 mL), a freshly prepared solution of sodium hydroxide (0.56 g, 2.0 mol/L in DI water) was slowly added and stirred at 298 K for 1 hour. The mixture was then added into a round-bottomed flask charged with CzDClT (3 g in 50 mL dry THF), and the solution was stirred and refluxed at 350 K for 1 hour. The crude solution was evaporated and extracted with dichloromethane three times. The resulting organic layer was dried using anhydrous sodium sulfate. After the solvent was removed by rotary evaporation, the residue was purified by flash column chromatography to give o-ITC (2.9 g, 68%) as a white solid. Before using, the white solid was recrystallized by a mixed solvent (chloroform/ethanol). 1   Gating strategies are referred to the method described in the BIO-RAD website (https://www.bio-radantibodies.com/flow-cytometry-gating-strategies.html). Gating was based on FSC/SCC together with fluorescent dyes and singlet populations. A forward-scatter (FSC) vs side-scatter (SSC) gate was used to gated on 4T1 cells to exclude debris. FSC-H vs FSC-A gate was used to gated on 4T1 singlet cells. The cell populations within the gate were analyzed based on the expression of markers.
Supplementary Fig. 11 | DNA damage evaluation in 4T1 single cell (a) and 4T1 cells (b) by comet assays. The radiotherapeutic effect was evaluated through comet assay. As can be seen, single cell electrophoresis (i.e., comet assay) showed that a nucleoid with a spherical shape existed in blank control group and ITC-NPs group, indicating no DNA migration. In both the radiotherapy (PBS+X-ray) and X-PDT (ITC-NPs+X-ray) groups, strand breaks were found to form a comet-like appearance. Compared to radiotherapy (PBS+X-ray), X-PDT (ITC-NPs+X-ray) group induced a higher frequency of strand breaks, and formed loose and bondless nucleoid. Supplementary Fig. 12 | Cell proliferation ability measured by clonogenic assay taken 14 days after radiotherapy (PBS+X-ray) or X-PDT (ITC-NPs+X-ray) treatments. The statistical data are expressed as mean values ± S.D. (n=3 independent experiments, **P = 0.0028). Statistical significance was assessed via unpaired two-sided Student t-test.