Novel near-infrared fluorescent integrin-targeted DFO analogue

Abstract

Desferrioxamine (DFO), a siderophore initially isolated from Streptomyces pilosus, possesses extraordinary metal binding properties with wide biomedical applications that include chelation therapy, nuclear imaging, and antiproliferation. In this work, we prepared a novel multifunctional agent consisting of (i) a near-infrared (NIR) fluorescent probe-cypate; (ii) an integrin alpha vbeta3 receptor (ABIR)-avid cyclic RGD peptide, and (iii) a DFO moiety, DFO-cypate-cyclo[RGDfK(approximately)] (1, with approximately representing the cypate conjugation site at the side chain of lysine; f is d-phenylalanine). Compound 1 and two control compounds, cypate-cyclo[RGDfK(approximately)] ( 2) and cypate-DFO ( 3), were synthesized by modular assembly of the corresponding protected RGD peptide cyclo[R(Pbf)GD(OBut)fK] and DFO on the dicarboxylic acid-containing cypate scaffold in solution. The three compounds exhibited similar UV-vis and emission spectral properties. Metal binding analysis shows that DFO as well as 1 and 3 exhibited relatively high binding affinity with Fe(III), Al(III), and Ga(III). In contrast to Ga(III), the binding of Fe to 1 and 3 quenched the fluorescence emission of cypate significantly, suggesting an efficient metal-mediated approach to perturb the spectral properties of NIR fluorescent carbocyanine probes. In vitro, 1 showed a high ABIR binding affinity (10 (-7) M) comparable to that of 2 and the reference peptide cyclo(RGDfV), indicating that both DFO and cypate motifs did not interfere significantly with the molecular recognition of the cyclic RGD motif with ABIR. Fluorescence microscopy showed that internalization of 1 and 2 in ABIR-positive A549 cells at 1 h postincubation was higher than 3 and cypate alone, demonstrating that incorporating ABIR-targeting RGD motif could improve cellular internalization of DFO analogues. The ensemble of these findings demonstrate the use of multifunctional NIR fluorescent ABIR-targeting DFO analogues to modulate the spectral properties of the NIR fluorescent probe by the chelating properties of DFO and visualize intracellular delivery of DFO by receptor-specific peptides. These features provide a strategy to explore the potential of 1 in tumor imaging and treatment as well as some molecular recognition processes mediated by metal ions.

Figure 1

Structures of DFO-cypate-[RGDfK(~)] (1) and the related compounds.

Figure 2

Normalized UV-Vis and emission spectra of DFO-cypate-[RGDfK(~)] (1), cypate-[RGDfK(~)] (2), and cypate-DFO (3) in DMSO.

Figure 3

ES-M spectra of 1 and its Ga(III), Al(III), and Fe(III) complexes (100uM) in 50% aq. ACN.

Figure 4

The relative binding affinities of 1and DFO with different metal ions at a molar ratio of 1:2 as determined by ES-MS.

Figure 5

Proposed metal complex structure of 1

Figure 6

Effects of Ga(III), Fe(III), and Cu(II) ions on UV-Vis (a, b, and c) and emission (d, e, and f) spectra of 1 in 20% aq. DMSO.

Figure 7

Internalization in A549 cells treated 10 h with 1 μM of compounds 3 (A), 2 (B) or 1 (C); quantitation of the internalization of each compound as a function of time (D).

Figure 8

Subcellular distribution of cells treated 10 h with 1 μM 3 (A), 2 (B) or 1 (C) in medium containing 0.02% DMSO co-stained with Mitotracker green; D, E, F are the inserts at 3x magnification corresponding to A, B, and C, respectively.

Figure 9

Subcellular distribution of cells treated 10 h with 3 (A), 2 (B) or 1 (C) co-stained with anti-LAMP1 antibody; D, E, F are the inserts at 3x magnification corresponding to A, B, and C, respectively.

Scheme 1

Synthesis of DFO-cypate-[RGDfK(~)] (1).