ROS-Responsive and pH-Sensitive Aminothiols Dual-Prodrug for Radiation Enteritis

Abstract

Radiation exposure can immediately trigger a burst of reactive oxygen species (ROS), which can induce severe cell death and long-term tissue damage. Therefore, instantaneous release of sufficient radioprotective drugs is vital to neutralize those accumulated ROS in IR-exposed areas. To achieve this goal, we designed, synthesized, and evaluated a novel oral ROS-responsive radioprotective compound (M1) with high biocompatibility and efficient ROS-scavenging ability to act as a promising oral drug for radiation protection. The compound is stably present in acidic environments and is hydrolyzed in the intestine to form active molecules rich in thiols. M1 can significantly remove cellular ROS and reduce DNA damage induced by γ-ray radiation. An in vivo experiment showed that oral administration of M1 effectively alleviates acute radiation-induced intestinal injury. Immunohistochemical staining showed that M1 improved cell proliferation, reduced cell apoptosis, and enhanced the epithelial integrity of intestinal crypts. This study provides a promising oral ROS-sensitive agent for acute intestinal radiation syndrome.

Keywords: ROS response; aminothiols; intestinal damage; oral administration; radioprotection.

Figure 1

(a) The synthesis of M1; (b) schematic illustration of ROS scavenging by M1 in vivo.

Figure 2

(a) The hydrolysis kinetics of M1 in solution in simulated gastric juice and simulated intestinal fluid; (b) ROS-responsive fragmentation of M1 molecules in PBS in vitro.

Figure 3

(a) The ROS levels in cells treated with various concentrations of M1 at 8 h after 4 Gy irradiation. (b) The ROS levels in M1 treated and untreated cells at 8 h after various doses of irradiation. (c) The ROS levels in M1 treated and untreated cells at various times after 4, 6, and 8 Gy irradiation. (d) Flow cytometry ROS analysis of HIEC-6 cells treated with groups of IR, IR + 0.1 mM, and IR + 0.5 mM at 24 h after 6 and 8 Gy irradiation (numbers represent ROS level). (e) Quantitative analysis of ROS level from (d). (f) Typical clone images of HIEC-6 cells belong to groups of IR, IR + 0.1 mM, and IR + 0.5 mM at 10 days after 0, 4, 6, 8, and 10 Gy irradiation. (g) Quantitative analysis of survival fractions from (f). Results are presented as the mean ± SD in triplicate and analyzed by one-way ANOVA.

Figure 4

(a) Flow cytometry apoptosis analysis of HIEC-6 cells treated with groups of IR, IR + Ami, IR + 0.1 mM, and IR + 0.5 mM at 24 h after 6 and 8 Gy irradiation. (b) Living cells analysis from (a). (c) Quantitative analysis of apoptosis cells from Figure 4a. Results are presented as the mean ± SD in triplicate.

Figure 5

(a) Typical comet assay images of HIEC-6 cells treated with groups of IR, IR + Ami, IR + 0.1 mM, and IR + 0.5 mM after 6 and 8 Gy irradiation. (b) Quantitative analysis of olive tail moment and tail DNA from (a). Results are expressed as the mean ± SD of 50 replicates. (c) CLSM microscopy observation of γ-H2AX immunofluorescence of HIEC-6 cells treated with groups of IR, IR + Ami, IR + 0.1 mM, and IR + 0.5 mM after 6 and 8 Gy irradiation. (d) Quantitative analysis of γ-H2AX foci from Figure 5c. Results are expressed as the mean ± SD of 20 replicates.

Figure 6

(a–c) Colon tissues, lengths, and hematopoietic system analyses of mice in control, IR, M1-500 mg/kg, IR + 125 mg/kg, IR + 250 mg/kg, IR + 500 mg/kg, and IR + Ami groups three days after 13 Gy ABI, n = 6 per group.

Figure 7

(a) Representative images showing the structure in cross-sections of the small intestine with hematoxylin and eosin (H&E), Ki67, and TUNEL staining. Scale bar: 200 μm. (b–e) Histogram showing the number of crypts, villus lengths, and Ki67 positive and TUNEL positive cells in intestinal sections from the control, IR, M1-500 mg/kg, IR + 125 mg/kg, IR + 250 mg/kg, IR + 500 mg/kg, and IR + Ami groups. The results are represented as mean ± SEM, n = 6 mice per group. *** p < 0.001, **** p < 0.0001 by one-way analysis of variance (ANOVA).