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

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 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.

Table 1 Size, PDI, and zeta potential of NPs

NPs	Size (nm)	PDI	Zeta potential (mV)
MNPs	43.2±3.7	0.157	−1.56±0.23
TMC–MNPs	55.1±4.3	0.249	+32.4±3.27
BN–TMC–MNPs	68.7±3.4	0.140	+36.8±4.41
DOTA–BN–TMC–MNPs	69.2±5.1	0.133	+ 16.8±1.86

Note: Values represent mean±SD, n=3.

Abbreviations: PDI, polydispersity index; NPs, nanoparticle; 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 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 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 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.