RGD-conjugated two-photon absorbing near-IR emitting fluorescent probes for tumor vasculature imaging

Abstract

Observation of the activation and inhibition of angiogenesis processes is important in the progression of cancer. Application of targeting peptides, such as a small peptide that contains adjacent L-arginine (R), glycine (G) and L-aspartic acid (D) residues can afford high selectivity and deep penetration in vessel imaging. To facilitate deep tissue vasculature imaging, probes that can be excited via two-photon absorption (2PA) in the near-infrared (NIR) and subsequently emit in the NIR are essential. In this study, the enhancement of tissue image quality with RGD conjugates was investigated with new NIR-emitting pyranyl fluorophore derivatives in two-photon fluorescence microscopy. Linear and nonlinear photophysical properties of the new probes were comprehensively characterized; significantly the probes exhibited good 2PA over a broad spectral range from 700-1100 nm. Cell and tissue images were then acquired and examined, revealing deep penetration and high contrast with the new pyranyl RGD-conjugates up to 350 μm in tumor tissue.

Figure 1

(A) Absorption (solid lines) and emission (dashed lines) spectra in toluene (black), DCM (red), and DMSO (blue), excitation anisotropy in silicone oil (dark green), and two-photon absorption spectrum in DCM (dark red circles) for 1; (B) absorption (solid lines) and emission (dashed lines) spectra in DMSO (black), and water (red), excitation anisotropy in DMSO (dark green), two-photon absorption spectrum in DMSO (dark red triangles) for 2; (C) absorption (solid lines) an emission (dashed lines) spectra in toluene (black), and DCM (red), excitation anisotropy in silicone oil (dark green), and two-photon absorption spectrum in DCM (dark blue triangles) for 3.

Figure 2

(A) Absorption and emission spectra for 1a in DMSO; (B) Absorption and emission spectra for 2a in DMSO; (C) Absorption (solid lines) and emission (dashed lines) spectra for 3 in organic nanoparticles without, 3a (black), and with, 3b (red) added lecithin.

Figure 3

Size distribution of organic nanoparticles 3a (A) and 3b (B) determined by DLS.

Figure 4

Fluorescence (A–C, E–G) and DIC overlay (D, H) images of U87MG cells after 1 h incubation with 1 (A), 1a (B–D), 2 (E) or 2a (F–H). B and F were pre-incubated with free RGD peptide. Scale bars are 10 μm.

Figure 5

Fluorescence (A–C) and DIC overlay (D) images of HCT116 cells after 1 h incubation with medium (A), 3a (B), or 3b (C, D). Scale bars are 10 μm.

Figure 6

3D reconstruction images show the vasculature in LLC tumor implants that were extracted from mice injected with 1 (A), 1a (B), 2 (C) and 2a (D). Arrows in A indicate non-specific uptake of 1. Each image shows 3D view (left), x-z view (right top) and x-y view (right bottom). Scale bars are 50 μm.

Figure 7

Optical cross-section fluorescence at 200 μm depth for 1 (A), 1a (B), 2 (D) and 2a (E) were analyzed. Scale bars are 50 μm.

Scheme 1

Synthesis of compounds 1 and 1a.

Scheme 2

Synthesis of compounds 2 and 2a.

Scheme 3

Synthesis of compounds 3.