IR808-ATIPA: A Dual-Function Agent for Enhanced Computed Tomography Imaging and Radiotherapy Sensitization in Cervical Cancer Treatment

Abstract

Radiotherapy is pivotal in localized cancer treatment, yet balancing therapeutic efficacy with collateral tissue damage remains challenging. Conventional iodinated contrast agents, limited by rapid metabolism and short imaging windows, hinder precise radiotherapy planning. We developed IR808-ATIPA, a tumor microenvironment-responsive iodine-based compound integrating computed tomography (CT) imaging and radiosensitization. Synthesized by covalently linking IR808 and ATIPA, IR808-ATIPA leverages iodine's x-ray attenuation for high-contrast imaging while enhancing radiation dose deposition in cervical cancer. Unlike conventional agents, its prolonged tumor retention improves imaging accuracy and therapeutic targeting. Evaluations in HeLa tumor-bearing nude mice demonstrated superior in vitro/in vivo imaging performance and sustained tumor accumulation. RNA sequencing revealed that IR808-ATIPA enhances radiotherapy efficacy by activating the ferroptosis pathway via increased reactive oxygen species production and amplified x-ray absorption. Safety assessments confirmed no notable toxicity to major organs. IR808-ATIPA functions dually as a CT contrast agent for precise tumor delineation and a radiosensitizer promoting ferroptosis-mediated radiotherapy enhancement. Its extended intratumoral retention enables targeted therapy, minimizing off-target effects. These findings highlight IR808-ATIPA as a promising theranostic agent, bridging imaging-guided precision and therapeutic efficacy to advance personalized cancer treatment.

Fig. 1.

Synthesis and characterization of IR808-ATIPA. (A) Chemical structure of iohexol, iodixanol, IR808, and ATIPA. (B) Photographs of IR808-ATIPA solutions at varying concentrations. (C) Physical properties of IR808-ATIPA in gram scale. (D) Synthetic route for IR808-ATIPA.

Fig. 2.

In vitro cellular damage assessment of IR808-ATIPA. (A and B) CCK-8 cytotoxicity assay results. (C and D) Colony formation assay results. (E and F) Flow cytometric analysis of cell apoptosis. (G) Immunofluorescence staining for γH2AX, indicating DNA damage (scale bar, 200 μm). Data are presented as mean ± SD (n = 3) and were analyzed by 2-way ANOVA with Bonferroni’s multiple comparison test. Significance levels: ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant (P > 0.05).

Fig. 3.

RNA-sequencing and DEG analysis in HeLa cells under various treatments. (A) Scatterplot showing the number of up- and down-regulated DEGs between groups. (B) Volcano plot comparing PBS group versus IR808-ATIPA + x-ray group. (C) Radar plot of the top 20 genes with the most significant differential expression between PBS and IR808-ATIPA + x-ray groups. (D) Bar chart of GO term enrichment for DEGs in the IR808-ATIPA + x-ray group. (E) Heatmap of DEGs related to apoptosis and ferroptosis across groups. (F) Circle plot of pathway enrichment. (G) Bar chart of KEGG pathway enrichment for DEGs in the IR808-ATIPA + x-ray group.

Fig. 4.

IR808-ATIPA treatment and x-ray irradiation induce DNA and morphological damage in HeLa cells. (A) BODIPY fluorescence images showing lipid droplet accumulation (scale bar, 200 μm). (B) Intracellular ROS levels detected by fluorescence (scale bar, 200 μm). (C) Quantification of intracellular GSH levels. (D) Measurement of intracellular GSH-PX levels. (E) Determination of intracellular MDA levels. (F) Western blot analysis of key proteins related to ferroptosis. (G) High-resolution transmission electron microscopy images of mitochondrial morphology (scale bar, 2 μm). Significance levels: ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant (P > 0.05).

Fig. 5.

CT imaging performance of IR808-ATIPA. (A and B) CT values and signal curve for varying concentrations of IR808-ATIPA. (C) Image of drug-injected tumor in a nude mouse. (D) Time-dependent CT value changes in nude mice after intratumoral injection of IR808-ATIPA. (E) CT images at different time points post-injection of IR808-ATIPA in HeLa tumor-bearing nude mice. Data are presented as mean ± SEM. Significance levels: ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant (P > 0.05).

Fig. 6.

Efficacy of IR808-ATIPA in in vivo radiotherapy. (A) Timeline of treatment. (B) Tumor growth curves during treatment. (C) Tumor weights at dissection. (D) Photographs of tumors at the end of treatment. (E) Body weight curves of mice during treatment. (F) H&E, GPX4, and Ki67 staining images of tumor tissues at treatment endpoint (scale bar, 200 μm). Data are presented as mean ± SD (n = 6). Significance levels: ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant (P > 0.05).

Fig. 7.

Biocompatibility assessment of IR808-ATIPA in vivo. Histological analysis of major organs post-treatment using H&E staining (scale bar, 200 μm).