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. 2021 May 14;13(10):2388.
doi: 10.3390/cancers13102388.

Synthesis and Characterization of Radiogallium-Labeled Cationic Amphiphilic Peptides as Tumor Imaging Agents

Takeshi Fuchigami  1 , Takeshi Chiga  1 , Sakura Yoshida  1 , Makoto Oba  2 , Yu Fukushima  1 , Hiromi Inoue  1 , Akari Matsuura  1 , Akira Toriba  1 , Morio Nakayama  1
Affiliations

Synthesis and Characterization of Radiogallium-Labeled Cationic Amphiphilic Peptides as Tumor Imaging Agents

Takeshi Fuchigami et al. Cancers (Basel). .

Abstract

SVS-1 is a cationic amphiphilic peptide (CAP) that exhibits a preferential cytotoxicity towards cancer cells over normal cells. In this study, we developed radiogallium-labeled SVS-1 (67Ga-NOTA-KV6), as well as two SVS-1 derivatives, with the repeating KV residues replaced by RV or HV (67Ga-NOTA-RV6 and 67Ga-NOTA-HV6). All three peptides showed high accumulation in epidermoid carcinoma KB cells (53-143% uptake/mg protein). Though 67Ga-NOTA-RV6 showed the highest uptake among the three CAPs, its uptake in 3T3-L1 fibroblasts was just as high, indicating a low selectivity. In contrast, the uptake of 67Ga-NOTA-KV6 and 67Ga-NOTA-HV6 into 3T3-L1 cells was significantly lower than that in KB cells. An endocytosis inhibition study suggested that the three 67Ga-NOTA-CAPs follow distinct pathways for internalization. In the biodistribution study, the tumor uptakes were found to be 4.46%, 4.76%, and 3.18% injected dose/g of tissue (% ID/g) for 67Ga-NOTA-KV6, 67Ga-NOTA-RV6, and 67Ga-NOTA-HV6, respectively, 30 min after administration. Though the radioactivity of these peptides in tumor tissue decreased gradually, 67Ga-NOTA-KV6, 67Ga-NOTA-RV6, and 67Ga-NOTA-HV6 reached high tumor/blood ratios (7.7, 8.0, and 3.8, respectively) and tumor/muscle ratios (5.0, 3.3, and 4.0, respectively) 120 min after administration. 67Ga-NOTA-HV6 showed a lower tumor uptake than the two other tracers, but it exhibited very low levels of uptake into peripheral organs. Overall, the replacement of lysine in SVS-1 with other basic amino acids significantly influenced its binding and internalization into cancer cells, as well as its in vivo pharmacokinetic profile. The high accessibility of these peptides to tumors and their ability to target the surface membranes of cancer cells make radiolabeled CAPs excellent candidates for use in tumor theranostics.

Keywords: cancer; cationic amphiphilic peptide; molecular imaging; radiogallium.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Design of fluorophore- and radiogallium chelator-conjugated cationic amphiphilic peptides (CAPs) as tumor imaging agents. Fluorescein- (A) and NOTA- (B) conjugated CAPs consisting of two segments of repeated cationic amino acid (lysine, arginine, or histidine) and valine residues joined by the β-turn tetrapeptide (−VDPPT−, where DP is d-proline), which allows the peptides to form folded β-hairpin structures on interaction with cancer cell membranes. Five peptide sequences were used as CAPs in this study (C).
Figure 2
Figure 2
Cellular uptake of FITC-CAPs into KB and 3T3-L1 cells at 2 h post-incubation. * p < 0.005, ** p < 0.0005, and *** p < 0.0001 (ANOVA and Tukey’s t test). Values are means ± SEM; n = 7–13.
Figure 3
Figure 3
Conforcal fluorescence imaging of FITC-CAPs in cell lines. KB (A–C) and 3T3-L1 (D–F) cells treated with 10 µM FITC-CAPs (green) for 1 h. Nuclei were stained by Hoechst 33342 (blue). Fluorescence images were taken with a confocal laser scanning microscope. Scale bar = 20 μm.
Figure 4
Figure 4
Zeta potential of liposomes composed of different PC/PS ratios in the presence or absence of NOTA-CAPs in a 10 mM HEPES buffer (pH 7.4). Liposomes were treated with 0, 10, or 20 µM of NOTA-KV6 (A), NOTA-RV6 (B), or NOTA-HV6 (C) for 1 h. Values are means ± SEM; n = 3.
Figure 5
Figure 5
CD spectra of NOTA-CAPs in the presence and absence of liposomes. CD spectra of 150 μM NOTA-KV6 (A), NOTA-RV6 (B), and NOTA-HV6 (C) in an aqueous buffer solution (50 mM BTP and 150 mM NaF at pH 7.4) (blue line) in the presence of neutral PC liposomes (yellow line) and in the presence of negatively charged DOPC/DOPS liposomes (1:1) (red line).
Figure 6
Figure 6
Cellular uptake of 67Ga-NOTA-KV6 (A), 67Ga-NOTA-RV6 (B), and 67Ga-NOTA-HV6 (C) in KB and 3T3-L1 cells. * p < 0.01 and ** p < 0.001 compared with 3T3-L1 cells. (ANOVA Bonferroni’s multiple comparison test). Values are mean ± SEM (n = 4–6).
Figure 7
Figure 7
Cellular uptake inhibition of 67Ga-NOTA-KV6 (A), 67Ga-NOTA-RV6 (B), and 67Ga-NOTA-HV6 (C) for 60 min in KB cells. Cells were incubated at 4 °C to investigate energy-dependent uptake. Three different inhibitors: a macropinocytosis inhibitor (EIPA), a caveolae inhibitor (nystatin), and a clathrin inhibitor (sucrose) were used to investigate the effects of inhibition of endocytosis. * p < 0.001 ** p < 0.005, and *** p < 0.0001 compared with control (ANOVA Dunnett’s multiple comparison test). Values are mean ± SEM (n = 12).
Figure 8
Figure 8
Schematic of the experimental design of the in vivo experiments (A): BALB/c nu/nu mice were subcutaneously injected with 1.0×107 KB cells (a). Tumors were allowed to reach 300–500 mm3 (1‒2 weeks after inoculation) prior to conducting bio-distribution studies (b). 67Ga-NOTA-CAPs (10 kBq/100 μL) were intravenously injected via the tail vein (c). Mice were sacrificed at each time point, and then blood samples were taken and organs were dissected. Tissues were then weighed and radioactivity was measured by automated gamma counting. In vivo biodistribution of 67Ga-NOTA-KV6 (B,E), 67Ga-NOTA-RV6 (C,F), and 67Ga-NOTA-HV6 (D,G) over time in KB tumor-bearing mice. Data are expressed as percent injected dose per gram of tissue (%ID/g). Each value represents the mean (standard deviation) of 4–6 mice. Tumor-to-blood and tumor-to-muscle ratios for 67Ga-NOTA-KV6 (H), 67Ga-NOTA-RV6 (I), and 67Ga-NOTA-HV6 (J) were calculated at different time points.
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