Image-guided tumor-selective radioiodine therapy of liver cancer after systemic nonviral delivery of the sodium iodide symporter gene

Abstract

We reported the induction of tumor-selective iodide uptake and therapeutic efficacy of (131)I in a hepatocellular carcinoma (HCC) xenograft mouse model, using novel polyplexes based on linear polyethylenimine (LPEI), shielded by polyethylene glycol (PEG), and coupled with the epidermal growth factor receptor-specific peptide GE11 (LPEI-PEG-GE11). The aim of the current study in the same HCC model was to evaluate the potential of biodegradable nanoparticle vectors based on pseudodendritic oligoamines (G2-HD-OEI) for systemic sodium iodide symporter (NIS) gene delivery and to compare efficiency and tumor specificity with LPEI-PEG-GE11. Transfection of HCC cells with NIS cDNA, using G2-HD-OEI, resulted in a 44-fold increase in iodide uptake in vitro as compared with a 22-fold increase using LPEI-PEG-GE11. After intravenous application of G2-HD-OEI/NIS HCC tumors accumulated 6-11% ID/g (123)I (percentage of the injected dose per gram tumor tissue) with an effective half-life of 10 hr (tumor-absorbed dose, 281 mGy/MBq) as measured by (123)I scintigraphic gamma camera or single-photon emission computed tomography computed tomography (SPECT CT) imaging, as compared with 6.5-9% ID/g with an effective half-life of only 6 hr (tumor-absorbed dose, 47 mGy/MBq) for LPEI-PEG-GE11. After only two cycles of G2-HD-OEI/NIS/(131)I application, a significant delay in tumor growth was observed with markedly improved survival. A similar degree of therapeutic efficacy had been observed after four cycles of LPEI-PEG-GE11/(131)I. These results clearly demonstrate that biodegradable nanoparticles based on OEI-grafted oligoamines show increased efficiency for systemic NIS gene transfer in an HCC model with similar tumor selectivity as compared with LPEI-PEG-GE11, and therefore represent a promising strategy for NIS-mediated radioiodine therapy of HCC.

FIG. 1.

(A) Iodide uptake activity of HuH7 cells after transfection with various conjugate/plasmid (c/p) ratios of G2-HD-OEI/NIS and LPEI-PEG-GE11/NIS. G2-HD-OEI/NIS showed the highest iodide uptake activity at a c/p ratio of 2 and LPEI-PEG-GE11/NIS at a c/p ratio of 0.8, respectively. (B) HuH7 cells transfected with G2-HD-OEI/NIS (c/p ratio, 2) showed a 44-fold increase in perchlorate-sensitive ¹²⁵I accumulation. In contrast, no perchlorate-sensitive iodide uptake above background level was observed in cells transfected with control vector or without DNA (***p<0.001).

FIG. 2.

¹²³I scans of nude mice harboring HuH7 tumors 4 hr after intraperitoneal injection of 18.5 MBq of ¹²³I after G2-HD-OEI-mediated NIS gene delivery (A). Whereas mice treated with control vectors (G2-HD-OEI/antisense-NIS) showed no tumoral radioiodine uptake (C), treatment with G2-HD-OEI/NIS induced significant tumor-specific iodide accumulation in HuH7 tumors (A), which was completely abolished on pretreatment with NaClO₄ (B). Radioiodine was also accumulated physiologically in thyroid, stomach, and bladder (A and C). Time course of ¹²³I accumulation in HuH7 tumors after systemic polyplex-mediated NIS gene delivery, using ¹²³I scintigraphy (D). Maximal tumoral radioiodine uptake was 6–11% ID/g for ¹²³I with an average effective t_1/2 of 10 hr for ¹³¹I.

FIG. 3.

¹²³I SPECT CT scan of tumor-bearing nude mice 5 hr after intraperitoneal injection of 50 MBq of ¹²³I after G2-HD-OEI-mediated NIS gene delivery (A) showed a tumor-specific iodide accumulation. In contrast, mice treated with control vectors (G2-HD-OEI/antisense-NIS) showed no significant tumoral radioiodine uptake (B). The color scale was adjusted so that the reference tissue had the same appearance in both images and the uptake in the tumors could be easily compared visually. Time course of tumoral iodide accumulation as determined by SPECT/CT scans (C). A maximal radioiodine uptake of 7% ID/g tumor was measured.

FIG. 4.

Evaluation of iodide biodistribution ex vivo after injection of 18.5 MBq of ¹²³I. Whereas tumors in NIS-transduced mice showed high perchlorate-sensitive iodide uptake activity (up to 6.6±1.95% ID/organ), nontarget organs revealed no significant radioiodine accumulation. No radioiodine accumulation was measured after injection of control vectors. Results are reported as the percentage of injected dose per organ±SD.

FIG. 5.

(A) Analysis of human NIS mRNA expression in HuH7 tumors and nontarget organs by qPCR. A significant level of NIS mRNA expression was induced in HuH7 tumors after systemic NIS gene transfer with or without sodium perchlorate pretreatment. Only a low background level of NIS mRNA expression was detected in untreated tumors, which was set as 1 arbitrary unit. Moreover, no significant NIS expression above the background level was found in tumors after application of G2-HD-OEI/antisense-NIS, or in nontarget organs, such as liver and lung. Results are reported as NIS-to-GAPDH ratios. (B and C) Immunohistochemical staining of HuH7 tumors 24 hr after G2-HD-OEI/NIS application showed clusters of primarily membrane-associated NIS-specific immunoreactivity (B, arrows). In contrast, HuH7 tumors treated with the control plasmid (G2-HD-OEI/antisense-NIS) did not reveal NIS-specific immunoreactivity (C). Original magnification,×200.

FIG. 6.

Radioiodine treatment of HuH7 tumors after systemic polyplex-mediated NIS gene transfer in vivo. Twenty-four hours after intravenous polyplex injection (long arrows), 55.5 MBq of ¹³¹I or saline was injected (short arrows). This treatment cycle was repeated once on days 7 and 8. ¹³¹I therapy after systemic G2-HD-OEI/NIS application resulted in a significant delay in tumor growth (A, *p<0.05), which was associated with markedly improved survival (B, Kaplan–Meier plot) as compared with the control groups that were injected with saline only, with G2-HD-OEI/NIS followed by saline application, or with G2-HD-OEI/antisense-NIS followed by saline or ¹³¹I.

FIG. 7.

Immunofluorescence analysis using a Ki67-specific antibody (green) and an antibody against CD31 (red, labeling blood vessels) showed significantly decreased proliferation and blood vessel density in NIS-transduced tumors (A and C) after ¹³¹I treatment as compared with mock-transduced tumors (B and D). Slides were counterstained with DAPI nuclear stain. Original magnification: (A and B)×100; (C and D)×200.