Development of Iodinated Indocyanine Green Analogs as a Strategy for Targeted Therapy of Liver Cancer

Abstract

Liver cancer is one of the leading causes of cancer-related deaths, with a significant increase in incidence worldwide. Novel therapies are needed to address this unmet clinical need. Indocyanine green (ICG) is a broadly used fluorescence-guided surgery (FGS) agent for liver tumor resection and has significant potential for conversion to a targeted therapy. Here, we report the design, synthesis, and investigation of a series of iodinated ICG analogs (I-ICG), which can be used to develop ICG-based targeted radiopharmaceutical therapy. We applied a CRISPR-based screen to identify the solute carrier transporter, OATP1B3, as a likely mechanism for ICG uptake. Our lead I-ICG compound specifically localizes to tumors in mice bearing liver cancer xenografts. This study introduces the chemistry needed to incorporate iodine onto the ICG scaffold and defines the impact of these modifications on key properties, including targeting liver cancer in vitro and in vivo.

Figure 1

(A) Synthesis of I-ICG compounds. (B) ICG and I-ICG compounds and their corresponding absorbances and emission. The properties shown include synthetic yield (%), absorbance maxima (λ_abs, nm)/emission maxima (λ_em, nm), extinction coefficient (σ, M^–1 cm^–1), and quantum yield (Φ_F, %) as measured in water. Reaction yields are indicated in parentheses.

Figure 2

Identification of putative ICG transporters using a SLC-CRISPRa screen. (A) Increase of the ICG (10 μM) fluorescence intensity after SLC-CRISPRa pool enrichment. The top 3% brightest population was sorted every week until the sorted population up to the 5th round achieved 99.99% enhancement. (B) Pie chart demonstrating the proportion of highly enriched sgRNAs in the unsorted SLC-CRISPRa pool and the 5th round enriched population. NGS count results are presented as percentages for the top five targets. (C) Gene expression patterns of the top five SLC genes in the 5th round enriched population. The fold change was normalized to the SLC-CRISPRa pool. Clones exhibited high ICG uptake, as demonstrated through (D) microscopy and (E) flow cytometry. Scale bar represents 200 μm.

Figure 3

(A) Western blot depicting the expression of the protein OATP1B3 or β-actin in HEK-293T cells, HEK-293T cells infected with the lentivirus OATP1c1.v1 (−, Lenti control, MOI 10), or HEK-293T cells infected with an OATP1B3 lentivirus (MOI 1–10). (B) Confocal fluorescence microscope images of cells incubated with a primary anti-OATP1B3 antibody overnight (4 °C) and then for 1 h with an AlexaFluor-488-goat-antimouse IgG (H+L) at room temperature. Cells were stained with DAPI and imaged in Hank’s buffered saline solution (HBSS) with a 63× oil-immersed lens. The full image is shown in Figure S6. (C) Flow cytometry of cells incubated with ICG (10 μM) for 1 h (at least three replicates per sample). Error bars represent the standard error of the mean. For statistical analysis, one-way ANOVA and Tukey’s multiple comparisons were performed (n = 3 or 4, F_3,11 = 7389, ****p ≤ 0.0001). (D) Mean fluorescence intensity of cells treated with 10 μM ICG or I-ICG compounds for 1 h.

Figure 4

(A) Fluorescent in vivo images of Hep3B-tumor-bearing mice treated with 4-I-ICG (2 mg/kg) and ICG (2 mg/kg) at 4, 24, and 48 h postinjection. Tumors are highlighted in red circles. (B) Ex vivo analysis of tumors (black) and livers (red) of Hep3B-tumor-bearing mice treated with 4-I-ICG (2 mg/kg) and ICG (2 mg/kg) at 48 h postinjection. Background is determined by the fluorescence signal from the neck. (C) Ex vivo tumor-to-liver ratios (TLRs) of Hep3B-tumor-bearing mice treated with 4-I-ICG and ICG at 48 h postinjection. (D) Fluorescent ex vivo images of tumors, livers, and lungs of HB52-, HB66-, or non-tumor-bearing mice treated with 4-I-ICG (10 mg/kg) at 72 h postinjection. (E) Ex vivo analysis of lungs, liver, and tumor of HB52-, HB66-, or non-tumor-bearing mice treated with 4-I-ICG (10 mg/kg) at 72 h postinjection, n = 1. Data points are displayed as mean ± SD, and the p-values were evaluated by the Student’s t test. *p ≤ 0.05, ***p ≤ 0.001, ns is nonsignificant.