The self-assembly of monosubstituted BODIPY and HFBI-RGD

Abstract

A novel fluorescent probe was constructed by the self-assembly of monosubstituted BODIPY and a novel targeted hydrophobin named hereafter as HFBI-RGD. Optical measurements and theoretical calculations confirmed that the spectral properties of the probe were greatly influenced by the BODIPY structure, the appropriate volume of BODIPY and the cavity of HFBI-RGD. The experiments in vivo and ex vivo demonstrated that the probe had excellent ability for tumor labelling.

Fig. 1. Three-dimensional structure of HFBI. The green part on the surface of HFBI is the hydrophobic patch that can bind to the hydrophobic solid surface.

Scheme 1. Synthesis of three mono-substituted BODIPYs.

Fig. 2. Fluorescence spectra of dyes 3a, 3b and 3c in CH₂Cl₂ with different concentrations. Condition: excitation wavelength 560 nm.

Fig. 3. SEM images of BODIPY, HFBI-RGD and HFBI-RGD/BODIPY. (a) BODIPY. The images from the left to right indicate the shapes of dyes 3a–3c, respectively. (b) HFBI-RGD. The figures from the left to right exhibit the overall, single, membrane of hydrophobic protein modified by RGD. (c) HFBI-RGD/BODIPY. The images from the left to right indicate the shapes of HFBI-RGD/3a–3c, respectively.

Fig. 4. Fluorescence spectra of dyes in the presence of different concentrations of HFBI-RGD aqueous solutions. (a) HFBI-RGD/3a. (b) HFBI-RGD/3b. Inset: the image of HFBI-RGD/3b upon the irradiation of ultraviolet lamp (365 nm) after two weeks. (c) HFBI-RGD/3c.

Fig. 5. Cytotoxicity of HFBI-RGD/BODIPY fluorescent probe by MTT assay. Black, cytotoxicity of HFBI-RGD/3a; red, cytotoxicity of HFBI-RGD/3b; blue, cytotoxicity of HFBI-RGD/3c. (n = 3, *P < 0.05).

Fig. 6. Confocal fluorescence images (40×) of glioma cells U-87 incubated with HFBI/3a or HFBI-RGD/3a, HFBI/3b or HFBI-RGD/3b, HFBI/3c or HFBI-RGD/3c (5 μM) for 4 h at 37 °C. Merged images, probe (red), cell nuclei (blue) are shown. (a) U-87 cells were incubated with HFBI/3a or HFBI-RGD/3a (5 μM) for 4 h; (b) U-87 cells were incubated with HFBI/3b or HFBI-RGD/3b (5 μM) for 4 h; (c) U-87 cells were incubated with HFBI/3c or HFBI-RGD/3c (5 μM) for 4 h. The excitation wavelengths of DAPI and HFBI-RGD/BODIPY fluorescent probe were 405 nm and 560 nm, respectively.

Fig. 7. (a) Time-dependent in vivo fluorescence images of nude mice bearing glioma cells U-87 after tail intravenous injection. Images were taken at 2 h, 4 h, 6 h, 8 h, 24 h, 48 h, and 72 h. (b) Fluorescence images of the main organs (heart, liver, spleen, lung, and kidneys) and tumors from U-87 tumor-bearing mice at different time intervals (24 h, 48 h, and 72 h).