Fluorinated diselenide nanoparticles for radiosensitizing therapy of cancer

Abstract

Radiation resistance of cancer cells represents one of the major challenges in cancer treatment. The novel self-assembled fluoralkylated diselenide nanoparticles (fluorosomes) based on seleno-l-cystine (17FSe₂) possess redox-active properties that autocatalytically decompose hydrogen peroxide (H₂O₂) and oxidize the intracellular glutathione (GSH) that results in regulation of cellular oxidative stress. Alkylfluorinated diselenide nanoparticles showed a significant cytotoxic and radiosensitizing effect on cancer cells. The EL-4 tumor-bearing C56BL/6 mice treated with 17FSe₂ followed by fractionated radiation treatment (4 × 2Gy) completely suppressed tumor growth. Our results suggest that described diselenide system behaves as a potent radiosensitizer agent targeting tumor growth and preventing tumor recurrence.

Keywords: Diselenide; Fluorosomes; Hypoxia; Radiosensitizers; Redox systems; Self-assembly nanoparticles.

Fig. 1.

DLS and cryo-/TEM measurements of self-assembly properties of 17FSe₂. (A) The radius of nanoparticles solubilized in PBS. (B) Change in the radius of nanoparticles during the first H₂O₂ addition. The blue line represents the point when H₂O₂ was added to the solution. (C) The radius change during additional addition of H₂O₂. (D) TEM microphotography of nontreated 17FSe₂ nanoparticles. (E) TEM microphotography of newly formed nanoparticles (fluorosomes) after H₂O₂ treatment. (F) Cryo-TEM microphotography of newly formed selenium fluorosome after H₂O₂ treatment. Red arrows are showing fluorinated phase bilayer. (G) Representation of possible nanoparticle transition and fluorosome formation in the presence of ROS.

Fig. 2.

¹H NMR and ⁷⁷Se NMR spectra of 17FSe₂ measured before (A, B) and after (C, D) the addition of H₂O₂ recorded in MeOD at 295 K. (E) Proposed structural changes of 17FSe₂ interacting with H₂O₂. (F–H) Chemiluminescence measurement of the decomposition of H₂O₂ via diselenide nanoparticles. The chemiluminescence signal was generated by 10 mM H₂O₂ and luminol with (red markers) and without (black markers) in the presence of 17FSe₂ (0.25 mM); 0.1 mM (F), 1 mM (G) and 20 mM (H) H₂O₂. (I) Heat flow was generated during ITC measurements between 10 × molar excess H₂O₂ in PBS and 17FSe₂ (0.25 mM) at various temperatures. (J-L) The generation of superoxide radicals in the presence of GSH detected by a lucigenin. Generation of superoxide by 17Fse₂ (J) and (K) seleno-l-cystine (boughs, 100 μL, 0.25 mM). (L) Comparison of the CL signal between equimolar solutions of 17FSe₂ and seleno-l-cystine (0.25 mM). (M) The record of the ITC measurement. Heat flow was generated during the oxidation of 17FSe₂ with a 4x molar excess of GSH at 37°C. The ability to generate superoxide radicals was measured in air-equilibrated aqueous solutions of GSH. (O) Proposed mechanism of the diselenides effect and structural changes during the ROS presence.

Fig. 3.

Cell viability assay and internalization of 17FSe₂ into cells. (A) Comparison of cell viability between the human breast adenocarcinoma cell line MDA-MB-231 (hypoxic 2% O₂/normoxic 21% O₂) and human fibroblasts after 40 h of treatment. Viability data are presented as the mean ± SE (n = 3); *P < 0.05, **P< 0.01. (B) Confocal image of the internalization of 17FSe₂/17FSe₂-Dy-505 in MDA-MB-231 cells after 24 h (upper left) and 48 h (all others). (C) Determination of total GSH level in cells cultivated under 2% oxygen for 40 h. The data are presented as the mean ± SE (n = 4, N = 2*10⁶ cells); *P < 0.01, **P < 0.001. (D) Generation of intracellular ROS determined by DCFH-DA after cell irradiation. Fluorescence signal generated by ROS in normoxic cancer cells treated with 17FSe₂ (). Fluorescence signal generated by ROS in hypoxic cell lines treated with 17FSe₂ (). The values are expressed as the mean ± SE; n = 3; significance levels are expressed as a comparison with the control group (normoxic-asterix, and hypoxic-circle). For normoxic cells, we found significant differences at the level *, P < 0.05 irradiated with 4 Gy (10 μM and 50 μM), and **, P < 0.001 for (100 μM) of 17FSe₂. Significant differences at the level **, P < 0.001 for cells irradiated with 2 Gy for (10 μM, 50 μM and 100 μM) of 17FSe₂. In the case of hypoxic cells, the significant differences were found only for non-irradiated cells ◦, P < 0.05 for 50 μM 17FSe₂ and ◦ ◦, P < 0.001 for 10 μM and 100 μM of 17FSe₂. (E) Experimental setup of clonogenic experiments (F) Clonogenic cell survival curves of cancer cells exposed to X-ray (0–8 Gy) after short oxygen pretreatment. One-way ANOVA followed by Tukey’s post-hoc test was applied in all significant level calculations.

Fig. 4.

The comparison of tumor volume growth during the treatment. (A) Non-irradiated control group (CO). (B) The representative tumor volume of the control group (CO). (C) Non-irradiated diselenide-treated group (17FSe₂). (D) The representative tumor volume of the Non-irradiated diselenide-treated group (17FSe₂). (E) Irradiated control (IR CO). (F) The representative tumor volume of the irradiated control group (IR CO). (G) Irradiated diselenide-treated group (IR 17FSe₂). (H) The representative tumor volume of the irradiated diselenide-treated group (IR 17FSe₂).