Dual-functional protein for one-step production of a soluble and targeted fluorescent dye

Abstract

Low water solubility and poor selectivity are two fundamental limitations that compromise applications of near-infrared (NIR) fluorescent probes. Methods: Here, a simple strategy that can resolve these problems simultaneously was developed by using a novel hybrid protein named RGD-HFBI that is produced by fusion of hydrophobin HFBI and arginine-glycine-aspartic acid (RGD) peptide. This unique hybrid protein inherits self-assembly and targeting functions from HFBI and RGD peptide respectively. Results: Boron-dipyrromethene (BODIPY) used as a model NIR dye can be efficiently dispersed in the RGD-HFBI solution by simple mixing and sonication for 30 min. The data shows that self-assembled RGD-HFBI forms a protein nanocage by using the BODIPY as the assembly template. Cell uptake assay proves that RGD-HFBI/BODIPY can efficiently stain αvβ3 integrin-positive cancer cells. Finally, in vivo affinity tests fully demonstrate that the soluble RGD-HFBI/BODIPY complex selectively targets and labels tumor sites of tumor-bearing mice due to the high selectivity of the RGD peptide. Conclusion: Our one-step strategy using dual-functional RGD-HFBI opens a novel route to generate soluble and targeted NIR fluorescent dyes in a very simple and efficient way and may be developed as a general strategy to broaden their applications.

Keywords: NIR fluorescent probes; RGD peptide; protein nanocage; self-assembly.

Figure 1

(A) X-ray crystal structure of the amphiphilic hydrophobin HFBI [PDB ID 2FZ6]. The sun-yellow part in the surface of HFBI represents the hydrophobic patch that is the basis for binding to various hydrophobic surfaces by strong hydrophobic interactions. (B) A RGD motif is linked with the N terminus of HFBI protein via a flexible linker and the resultant hybrid protein was named RGD-HFBI, which owns dual functions based on its structural properties. The first function derived from the HFBI part is the ability to self-assemble and bind onto different hydrophobic surfaces. The second function inherited from the RGD motif is to facilitate recognition and binging to the target αvβ3 integrin protein on the surface of tumor angiogenic endothelial cells. (C) Purification results of RGD-HFBI and native HFBI with an RP-HPLC system. (D) Tricine-SDS-PAGE results of the purified RGD-HFBI and native HFBI. (E) Self-assembled RGD-HFBI film and native HFBI film on TEM copper grids. (F) WCA measurements of polystyrene and mica before and after modification with RGD-HFBI and native HFBI.

Figure 2

(A) Synthetic procedure for the synthesis of the BODIPY derivative. (B-C) Absorption and fluorescence spectra of the BODIPY dye at different concentrations (10, 20, 30 and 40 μM) in DMSO. (D) Photophysical properties of the BODIPY dye.

Figure 3

(A-B) Absorption and fluorescence spectra of the BODIPY dye solubilized in RGD-HFBI and native HFBI solution at different concentrations (0.05, 0.1, 0.15 and 0.2 mg/mL). (C) Stability of RGD-HFBI- and native HFBI-dispersed BODIPY solution over 20 days. (D) Particles size of RGD-HFBI- and native HFBI-treated BODIPY dye.

Figure 4

(A) TEM images of BODIPY dissolved in different solvents (H₂O, DMSO, HFBI and RGD-HFBI). The red dashed line represents the RGD-HFBI shell or film. (B) Atomic force microscopy images of self-assembled RGD-HFBI (native HFBI) film on a mica surface. (C) Wide XPS spectra of RGD-HFBI/BODIPY, HFBI/BODIPY and BODIPY, and high-resolution spectra of F1s, B1s and O1s. (D) FTIR spectra of RGD-HFBI/BODIPY, HFBI/BODIPY and BODIPY.

Figure 5

(A) MTT assay results of three different cancer cell lines including U-87MG (high αvβ3 integrin expression), HeLa (low αvβ3 integrin expression) and MCF-7 cells (without αvβ3 integrin expression). Those three cell lines were treated with different concentrations (0, 0.1, 1, 5, 10, 25, 50, 100, 250 and 500 µM) of RGD-HFBI/BODIPY, HFBI/BODIPY and BODIPY for 48 h. (B) Flow cytometry analysis of U-87MG, Hela and MCF-7 cells incubated with RGD-HFBI/BODIPY, HFBI/BODIPY and BODIPY for 4 h. The concentration of all probes was 10 μM and cells without treatments were used as the blank controls (red lines). (C) Confocal microscopy images of U-87MG, Hela and MCF-7 cells treated with RGD-HFBI/BODIPY, HFBI/BODIPY, BODIPY and synthetic RGD peptide.

Figure 6

(A) NIR fluorescence images of nude mice bearing U-87MG tumor after intravenous injection of RGD-HFBI/BODIPY and HFBI/BODIPY. (B) The ex vivo optical images of organs (heart, liver, spleen, lung and kidney) and tumors of the U-87MG tumor-bearing mice sacrificed at three time points (24, 48 and 72 h) after administration. (C) Quantitation of in vivo fluorescence intensity of U-87MG tumor after administration of RGD-HFBI/BODIPY and HFBI/BODIPY in the tumor-bearing mice at different time points (2, 4, 6, 8, 24, 48 and 72 h). (D) Quantitation of ex vivo fluorescence intensity of harvested tumors of the U-87MG tumor-bearing mice sacrificed at three time points (24, 48 and 72 h) after administration. (E) NIR fluorescence images of nude mice bearing HeLa tumor after intravenous injection of RGD-HFBI/BODIPY and HFBI/BODIPY. (F) The ex vivo optical images of organs (heart, liver, spleen, lung and kidney) and tumors of the HeLa tumor-bearing mice sacrificed at three time points (24, 48 and 72 h) after administration. (G) Quantitation of in vivo fluorescence intensity of HeLa tumor after administration of RGD-HFBI/BODIPY and HFBI/BODIPY in the tumor-bearing mice at different time points (2, 4, 6, 8, 24, 48 and 72 h). (H) Quantitation of in vitro fluorescence intensity of harvested tumors of the HeLa tumor-bearing mice sacrificed at three time points (24, 48 and 72 h) after administration.