Biological activity of gold nanoparticles combined with the NFL-TBS.40-63 peptide, or with other cell penetrating peptides, on rat glioblastoma cells

Abstract

Targeting, detecting, and destroying selectively cancer cells or specific organelles is a major challenge of nanomedicine. Recently, a new methodology was conceived to synthesize gold nanoparticles combined with a peptide having a C-terminal biotin (BIOT-NFL-peptide). This methodology called "Method IN" allows specific interactions between the BIOT-NFL-peptide, the polyethylene glycol diacid (PEG-COOH) and the gold salt (Au III) to produce multifunctional hybrid nano-carriers called BIOT-NFL-PEG-AuNPs. Here, we show that it is possible to use this strategy to synthesize multifunctional hybrid nano-carriers with other cell-penetrating peptides including TAT and Vim-peptides. Ex-vivo studies on F98 rat glioblastoma cells show that these new nanovectors acquire the cellular entry function of peptides and the gold particles make it possible to visualize by electron microscopy their localization in organelles. Thus, these new multifunctional nanovectors offer promising possibilities for the theranostic field, including the cell-penetrating property of the peptide, the intra-organelle localization of gold particles and their possible thermoplasmonic properties, as well as the stealth property of PEG.

Keywords: AuNPs, Gold nanoparticles; BIOT-CPP-PEG-AuNPs, Biotinylated cell-penetrating peptides polyethylene glycol gold nanoparticles; BIOT-NFL-PEG-AuNPs, Biotinylated NFL-peptide polyethylene glycol gold nanoparticles; BIOT-NFL-peptide; BIOT-TAT-PEG-AuNPs, Biotinylated TAT-peptide polyethylene glycol gold nanoparticles; BIOT-TAT-peptide; BIOT-Vim-PEG-AuNPs, Biotinylated Vim-peptide polyethylene glycol gold nanoparticles; BIOT-Vim-peptide; CPP, Cell-penetrating peptides; GBM, Glioblastoma; Gold nanoparticles; NFL-peptide, NFL-TBS.40-63, or Neuro Filament Low subunit Tubulin Binding Site 40-63; PEG-AuNPs, Polyethylene glycol gold nanoparticles; Rat glioblastoma cells; TBS, Tubulin Binding Sites; TEM, Transmission electron microscopy; Transmission electron microscopy; Vim-peptide, Vim-TBS.58-81, or Vimentin Tubulin Binding Site 58-81.

Graphical abstract

Fig. 1

Schematic representation of synthesis of gold nanoparticles combined with the BIOT-NFL-peptide (BIOT-NFL-PEG-AuNPs). As described in Arib et al., 2022, a complex between tetrachloroauric acid (HAuCl₄) with BIOT-NFL-peptide was created. Then, a stabilizing agent, dicarboxylic polyethylene glycol-600 (PEG(COOH)₂), and sodium borohydride (NaBH₄) were added. Finally, BIOT-NFL-PEG-AuNPs were synthetized. The same process was used for BIOT-TAT-PEG-AuNPs, and BIOT-Vim-PEG-AuNPs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Characterization of gold nanoparticles (PEG-AuNPs) combined or not with the BIOT-CPP-peptide by transmission electron microscopy (TEM). (A) TEM images of PEG-AuNPs, (B) of BIOT-NFL-PEG-AuNPs, (C) of BIOT-TAT-PEG-AuNPs, and (D) of BIOT-Vim-PEG-AuNPs. Scale bars: 50 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

In vitro effects of gold nanoparticles (PEG-AuNPs) alone, or with the BIOT-NFL-peptide (BIOT-NFL-PEG-AuNPs) on rat glioblastoma cells (F98) mitochondrial activity. F98 cells were treated with nanoparticles alone, with the BIOT-NFL-PEG-AuNPs at 0, 50, 100, 250, 500 or 1000 μmol/L; or with the positive control Colchicine (Col, 1 μg/mL), for 24 h (A) or 72 h (B). Then, the mitochondrial activity was evaluated by MTS assay. Experiments were performed at least in triplicate. Data are represented as mean ± SEM. Statistical analysis was performed with Student's t-test (*p ˂ 0.05; **p ˂ 0.005 and ***p ˂ 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

In vitro effects of gold nanoparticles (PEG-AuNPs) alone, or with the BIOT-TAT-peptide (BIOT-TAT-PEG-AuNPs), or with the BIOT-Vim-peptide (BIOT-Vim-PEG-AuNPs) on rat glioblastoma cells (F98) mitochondrial activity. F98 cells were treated with nanoparticles alone, with the different CPP-PEG-AuNPs nanoparticles at 0, 50, 100, 250, 500 or 1000 μmol/L; or with the positive control Colchicine (Col, 1 μg/mL), for 24 h (A) or 72 h (B). Then, the mitochondrial activity was evaluated by MTS assay. Experiments were performed at least in triplicate. # No cells are present at the end of the treatment incubation. Data are represented as mean ± SEM. Statistical analysis was performed with Student's t-test (*p ˂ 0.05 and ***p ˂ 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Transmission electron microscope images illustrating the internalization of gold nanoparticles (PEG-AuNPs) alone or coupled with the BIOT-NFL-peptide (BIOT-NFL-PEG-AuNPs) in rat glioblastoma cells. F98 cells were treated with nanoparticles at 500 μmol/L for 72 h. Cells were observed with a TEM. (A) Control F98 cells (no treatment), (B) F98 cells treated with PEG-AuNPs, and (C) F98 cells treated with BIOT-NFL-PEG-AuNPs. N for nucleus and V for vacuoles. Scale bars (at left): 2 μm. Scale bars (at right): 200 nm for A and 50 nm for B and C. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Transmission electron microscope images illustrating the internalization of gold nanoparticles (PEG-AuNPs) alone, coupled with the BIOT-TAT-peptide (BIOT-TAT-PEG-AuNPs), or with the BIOT-Vim-peptide (BIOT-Vim-PEG-AuNPs) in rat glioblastoma cells. F98 cells were treated with nanoparticles at 250 μmol/L for 24 h. Cells were observed with a TEM. (A) F98 cells treated with PEG-AuNPs, (B) F98 cells treated with BIOT-TAT-PEG-AuNPs, and (C) F98 cells treated with BIOT-Vim-PEG-AuNPs. N for nucleus and V for vacuoles. Scale bars (at left): 2 μm. Scale bars (at right): 50 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Biological TEM images showing the morphology of PEG-AuNPs alone and combined with BIOT-CPP-peptides after F98 cells treatment. F98 cells were treated with 500 μmol/L of PEG-AuNPs (A), or of BIOT-NFL-PEG-AuNPs (B) for 72 h. F98 cells were treated with 250 μmol/L of BIOT-TAT-PEG-AuNPs (C), or of BIOT-Vim-PEG-AuNPs (D) for 24 h. Scale bars: 50 nm.