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Original Research

68Ga-radiolabeled bombesin-conjugated to trimethyl chitosan-coated superparamagnetic nanoparticles for molecular imaging: preparation, characterization and biological evaluation

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Pages 2591-2605 | Published online: 10 Apr 2019

Figures & data

Figure 1 Schematic illustration of the synthesis and radiolabeling of DOTA-BN-TMC-MNPs.

Abbreviations: DOTA, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid; BN, bombesin; TMC, N,N,N-trimethyl chitosan; MNP, magnetic nanoparticle; NMP, N-methyl-2-pyrrolidone.

Figure 1 Schematic illustration of the synthesis and radiolabeling of DOTA-BN-TMC-MNPs.Abbreviations: DOTA, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid; BN, bombesin; TMC, N,N,N-trimethyl chitosan; MNP, magnetic nanoparticle; NMP, N-methyl-2-pyrrolidone.

Figure 2 Photographs of (A) MNPs, (B) TMC–MNPs, (C) BN–TMC–MNPs, (D) DOTA–BN–TMC–MNPs, and (E) 68Ga–DOTA–BN–TMC–MNPs in suspension. TMC–MNPs, BN–TMC–MNPs, DOTA–BN–TMC–MNPs, and 68Ga–DOTA–BN–TMC–MNPs showed dispersion stability in suspension, but MNPs (A) precipitated quickly.

Abbreviations: MNP, magnetic nanoparticle; TMC, N,N,N-trimethyl chitosan; BN, bombesin; DOTA, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid; 68Ga, gallium-68.

Figure 2 Photographs of (A) MNPs, (B) TMC–MNPs, (C) BN–TMC–MNPs, (D) DOTA–BN–TMC–MNPs, and (E) 68Ga–DOTA–BN–TMC–MNPs in suspension. TMC–MNPs, BN–TMC–MNPs, DOTA–BN–TMC–MNPs, and 68Ga–DOTA–BN–TMC–MNPs showed dispersion stability in suspension, but MNPs (A) precipitated quickly.Abbreviations: MNP, magnetic nanoparticle; TMC, N,N,N-trimethyl chitosan; BN, bombesin; DOTA, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid; 68Ga, gallium-68.

Figure 3 Characterization of MNPs and TMC–MNPs using PXRD, VSM, and TGA. (A) PXRD patterns of MNPs and TMC–MNPs. (B) TGA curves of MNPs and TMC–MNPs. (C) Hysteresis curves of MNPs and TMC–MNPs.

Abbreviations: H, magnetic field; M, magnetization; MNP, magnetic nanoparticle; TMC, N,N,N-trimethyl chitosan; PXRD, Powder X-ray diffraction; VSM, vibrating sample magnetometer; TGA, thermogravimetric analysis.

Figure 3 Characterization of MNPs and TMC–MNPs using PXRD, VSM, and TGA. (A) PXRD patterns of MNPs and TMC–MNPs. (B) TGA curves of MNPs and TMC–MNPs. (C) Hysteresis curves of MNPs and TMC–MNPs.Abbreviations: H, magnetic field; M, magnetization; MNP, magnetic nanoparticle; TMC, N,N,N-trimethyl chitosan; PXRD, Powder X-ray diffraction; VSM, vibrating sample magnetometer; TGA, thermogravimetric analysis.

Table 1 Size, PDI, and zeta potential of NPs

Figure 4 FTIR spectra of (A) MNPs, (B) TMC, (C) TMC–MNPs, (D) BN–TMC–MNPs, and (E) DOTA–BN–TMC–MNPs.

Abbreviations: FTIR, Fourier transform infrared; MNP, magnetic nanoparticle; TMC, N,N,N-trimethyl chitosan; BN, bombesin; DOTA, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid.

Figure 4 FTIR spectra of (A) MNPs, (B) TMC, (C) TMC–MNPs, (D) BN–TMC–MNPs, and (E) DOTA–BN–TMC–MNPs.Abbreviations: FTIR, Fourier transform infrared; MNP, magnetic nanoparticle; TMC, N,N,N-trimethyl chitosan; BN, bombesin; DOTA, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid.

Figure 5 TEM images of (A) MNPs, (B) TMC–MNPs, (C) BN–TMC–MNPs, and (D) DOTA–BN–TMC–MNPs.

Abbreviations: TEM, transmission electron microscopy; MNP, magnetic nanoparticle; TMC, N,N,N-trimethyl chitosan; BN, bombesin; DOTA, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid; SPION, superparamagnetic iron oxide nanoparticle.

Figure 5 TEM images of (A) MNPs, (B) TMC–MNPs, (C) BN–TMC–MNPs, and (D) DOTA–BN–TMC–MNPs.Abbreviations: TEM, transmission electron microscopy; MNP, magnetic nanoparticle; TMC, N,N,N-trimethyl chitosan; BN, bombesin; DOTA, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid; SPION, superparamagnetic iron oxide nanoparticle.

Figure 6 RCP of 68Ga–DOTA–BN–TMC–MNPs in human serum at 37°C. Values represent the mean±SD, n=3.

Abbreviations: RCP, radiochemical purity; 68Ga, gallium-68; DOTA, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid; BN, bombesin; TMC, N,N,N-trimethyl chitosan; MNP, magnetic nanoparticle.

Figure 6 RCP of 68Ga–DOTA–BN–TMC–MNPs in human serum at 37°C. Values represent the mean±SD, n=3.Abbreviations: RCP, radiochemical purity; 68Ga, gallium-68; DOTA, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid; BN, bombesin; TMC, N,N,N-trimethyl chitosan; MNP, magnetic nanoparticle.

Figure 7 MTT assay of DOTA–BN–TMC–MNPs on T-47D cells after 24 and 48 hours.

Abbreviations: DOTA, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclodo-decane tetraacetic acid; BN, bombesin; TMC, N,N,N-trimethyl chitosan; MNP, magnetic nanoparticle.

Figure 7 MTT assay of DOTA–BN–TMC–MNPs on T-47D cells after 24 and 48 hours.Abbreviations: DOTA, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclodo-decane tetraacetic acid; BN, bombesin; TMC, N,N,N-trimethyl chitosan; MNP, magnetic nanoparticle.

Figure 8 (A) Saturation binding study of 99mTc–HYNIC–GABA–BN (7–14) to GRP receptors of T-47D cells and (B) inhibition of 99mTc–HYNIC–GABA–BN (7–14) binding to T-47D cells with the various concentrations of DOTA–BN–TMC–MNPs. Results of a representative experiment are expressed as the percentage of radioactivity bound to the cells (mean±SD, n=3).

Abbreviations: 99mTc, Technetium-99m; HYNIC, hydrazinonicotinamide; GABA, gamma-aminobutyric acid; BN, bombesin; GRP, gastrin-releasing peptide; DOTA, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid; TMC, N,N,N-trimethyl chitosan; MNP, magnetic nanoparticle.

Figure 8 (A) Saturation binding study of 99mTc–HYNIC–GABA–BN (7–14) to GRP receptors of T-47D cells and (B) inhibition of 99mTc–HYNIC–GABA–BN (7–14) binding to T-47D cells with the various concentrations of DOTA–BN–TMC–MNPs. Results of a representative experiment are expressed as the percentage of radioactivity bound to the cells (mean±SD, n=3).Abbreviations: 99mTc, Technetium-99m; HYNIC, hydrazinonicotinamide; GABA, gamma-aminobutyric acid; BN, bombesin; GRP, gastrin-releasing peptide; DOTA, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid; TMC, N,N,N-trimethyl chitosan; MNP, magnetic nanoparticle.

Figure 9 (A) Biodistribution of 68Ga–DOTA–BN–TMC–MNPs in tumor-bearing mice at 30, 60, 90, and 120 minutes post-injection (ID%/g±SD, n=3) and (B) tumor-to-organ ratios at 30, 60, 90, and 120 minutes post-injection.

Abbreviations: %ID/g, percent injected dose per gram; 68Ga, gallium-68; DOTA, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid; BN, bombesin; TMC, N,N,N-trimethyl chitosan; MNP, magnetic nanoparticle.

Figure 9 (A) Biodistribution of 68Ga–DOTA–BN–TMC–MNPs in tumor-bearing mice at 30, 60, 90, and 120 minutes post-injection (ID%/g±SD, n=3) and (B) tumor-to-organ ratios at 30, 60, 90, and 120 minutes post-injection.Abbreviations: %ID/g, percent injected dose per gram; 68Ga, gallium-68; DOTA, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid; BN, bombesin; TMC, N,N,N-trimethyl chitosan; MNP, magnetic nanoparticle.

Figure 10 Phantom study, MRI, and PET/CT of DOTA–BN–TMC–MNPs and 68Ga–DOTA–BN–TMC–MNPs. (A) MRI phantom study of serially diluted aqueous solutions of 200 µM DOTA–BN–TMC–MNPs for Fe and (B) relaxation rate (1/T2) as a function of iron concentration. MR images of shoulder xenograft nude mouse (C) before and (D) after DOTA–BN–TMC–MNPs injection through the tail vein (1 hour) under the 3T magnetic field. Yellow circle demonstrates the uptake of NPs to the tumor. (E) PET/CT image of a nude mice bearing T-47D BC tumor in the right leg in the supine position following the injection of 3.7 MBq 68Ga–DOTA–BN–TMC–MNPs after 120 minutes.

Abbreviations: 1/T2, relaxation rate; MR, magnetic resonance; MRI, MR imaging; PET, positron emission tomography; CT, computed tomography; DOTA, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid; BN, bombesin; TMC, N,N,N-trimethyl chitosan; MNP, magnetic nanoparticle; 68Ga, gallium-68; NP, nanoparticle; BC, breast cancer.

Figure 10 Phantom study, MRI, and PET/CT of DOTA–BN–TMC–MNPs and 68Ga–DOTA–BN–TMC–MNPs. (A) MRI phantom study of serially diluted aqueous solutions of 200 µM DOTA–BN–TMC–MNPs for Fe and (B) relaxation rate (1/T2) as a function of iron concentration. MR images of shoulder xenograft nude mouse (C) before and (D) after DOTA–BN–TMC–MNPs injection through the tail vein (1 hour) under the 3T magnetic field. Yellow circle demonstrates the uptake of NPs to the tumor. (E) PET/CT image of a nude mice bearing T-47D BC tumor in the right leg in the supine position following the injection of 3.7 MBq 68Ga–DOTA–BN–TMC–MNPs after 120 minutes.Abbreviations: 1/T2, relaxation rate; MR, magnetic resonance; MRI, MR imaging; PET, positron emission tomography; CT, computed tomography; DOTA, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid; BN, bombesin; TMC, N,N,N-trimethyl chitosan; MNP, magnetic nanoparticle; 68Ga, gallium-68; NP, nanoparticle; BC, breast cancer.