Target Audience

Researchers, radiologists and other clinicians interested in tumor-targeted imaging, cancer therapy, and CEST imaging.

Purpose

We developed a new molecular probe termed “RRVR-Olsa” (Fig. 1a), in which the exchangeable hydroxyl protons in olsalazine provide chemical exchange saturation transfer (CEST) contrast with the cell-penetrating peptide RRVR acting as a specific substrate for furin¹. After the RRVR-Olsa enters furin-expressing cells, the peptide is cleaved by furin, initiating a condensation reaction between the GSH-induced 1,2-aminothiol group and the cyano group of the 2-cyanobenzothiazole motif^2-3, thereby resulting in the formation of olsalazine nanoparticles (Olsa-NPs) to enhance CEST signal. As olsalazine is an effective drug for colon cancer⁴, we investigate whether intracellular assembly of Olsa-NPs could represent a new theranostic approach.

Methods

CEST imaging: Furin-overexpressing HCT116 human colon cancer cells and furin-deficient (control) LoVo human colon cancer cells were incubated with or without 150 µM RRVR-Olsa for 24 h, and then cells were washed and collected in 5 mm NMR tubes. Cells were mixed with 2% agarose to prevent sedimentation. A modified RARE sequence (TR/TE=6,000/5 ms, RARE factor=32, 1 mm slice thickness, FOV=14×17 mm, matrix size=64×64, resolution=0.22×0.27 mm, NA=2, and a saturation pulse B₁=3.6 µT/4 s) was used. For in vivo tumor-targeting studies, each nude mouse (NU/J from Jackson, female, 6-8 weeks) was subcutaneously injected with 1x10⁶ LoVo cells in the left thigh and 1x10⁶ HCT116 cells in the right thigh. When the tumor reached a size of 5-8 mm, mice were injected i.v. with 0.1g/kg bw RRVR-Olsa, and CEST MRI was performed after 1.5 h on a 11.7 T Bruker horizontal scanner. A modified RARE sequence (TR/TE=5,000/3.7 ms, RARE factor=23, 1 mm slice thickness, FOV=3×3 cm, matrix size=64×64, resolution=0.48×0.48, NA=2 and a saturation pulse B₁=3.6 µT/3 s) was used. Cell viability: The number of cells was counted after incubation with RRVR-Olsa or olsalazine at different concentrations (125, 250, and 500 µM) for 48 h (HCT116 cells), after co-incubation of 250 µM RRVR-Olsa and furin inhibitor (HCT116 cells), and after incubation with 250 µM RRVR-Olsa (LoVo cells). Cell viability was calculated as a percentage of control (untreated) set at 100%. Data were expressed as means ± SD from three independent experiments.

Results

Fig. 1b clearly shows an enhanced CEST MRI contrast for furin-overexpressing HCT116 cells compared to furin-deficient LoVo cells and furin inhibitor-treated HCT116 cells, implying that the furin-catalyzed intracellular formation of Olsa-NPs is accountable for the signal enhancement. Cell toxicity was observed for incubation with 125 µM RRVR-Olsa, but not for free olsalazine that showed low cytotoxicity up to 500 µM. A comparable cytotoxicity of free olsalazine vs. RRVR-Olsa did not occur until the drug concentration was increased to 2.5 mM (Fig. 2). Furthermore, RRVR-Olsa exhibited much lower cell viability in HCT116 cells than in LoVo cells and the furin inhibitor-treated HCT116 cells, indicating that the cytotoxicity is induced by the furin-dependent self-assembly of Olsa-NPs. At 1.5 h after i.v. injection of RRVR-Olsa, furin-overexpressing HCT116 tumors and low furin-expressing LoVo tumors could be clearly distinguished from each other in vivo on CEST MRI (Fig. 3).

Discussion

After a condensation reaction, olsalazine nanoparticles are formed which accumulate inside furin-overexpressing HCT116 cells. Its prolonged olsalazine drug release properties enhances its cytotoxicity compared to free olsalazine. Through the specific intracellular interaction with furin, the accumulation of RRVR-Olsa enables in vivo CEST MRI differentiation between furin-overexpressing and furin low-expressing tumors. Hence, RRVR-Olsa may find applications as a new theranostic probe.

Acknowledgements

This project was supported by the Pearl and Yueh-Heng Yang Foundation.

References

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