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. 2020 Jun 21;10(6):1211.
doi: 10.3390/nano10061211.

Integrin-Targeting Dye-Doped PEG-Shell/Silica-Core Nanoparticles Mimicking the Proapoptotic Smac/DIABLO Protein

Rossella De Marco  1 , Enrico Rampazzo  2 , Junwei Zhao  2 , Luca Prodi  2 , Mayra Paolillo  3 , Pierre Picchetti  4 , Francesca Gallo  2 , Natalia Calonghi  5 , Luca Gentilucci  2
Affiliations

Integrin-Targeting Dye-Doped PEG-Shell/Silica-Core Nanoparticles Mimicking the Proapoptotic Smac/DIABLO Protein

Rossella De Marco et al. Nanomaterials (Basel). .

Abstract

Cancer cells demonstrate elevated expression levels of the inhibitor of apoptosis proteins (IAPs), contributing to tumor cell survival, disease progression, chemo-resistance, and poor prognosis. Smac/DIABLO is a mitochondrial protein that promotes apoptosis by neutralizing members of the IAP family. Herein, we describe the preparation and in vitro validation of a synthetic mimic of Smac/DIABLO, based on fluorescent polyethylene glycol (PEG)-coated silica-core nanoparticles (NPs) carrying a Smac/DIABLO-derived pro-apoptotic peptide and a tumor-homing integrin peptide ligand. At low μM concentration, the NPs showed significant toxicity towards A549, U373, and HeLa cancer cells and modest toxicity towards other integrin-expressing cells, correlated with integrin-mediated cell uptake and consequent highly increased levels of apoptotic activity, without perturbing cells not expressing the α5 integrin subunit.

Keywords: AVPI; IAP; RGD; Smac/DIABLO; cancer; cellular uptake; confocal microscopy; drug delivery; silica nanoparticles.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) Molecular components involved in the synthesis of nanoparticles (NPs)–N3 and functionalization scheme with the peptides H–Ala–Val–Pro–Ile (AVPI)–alkyne and/or cyclo Arg–Gly–Asp (cRGD)–alkyne for the preparation of AVPI–NPs, cRGD–NPs, and AVPI/cRGD–NPs. Morphological characterization of NPs–N3: (B) TEM images of NP–N3 (scale bar = 100 nm) and (C) TEM distribution of the diameters (nm). (D) Hydrodynamic diameters distribution by volume of NP–N3 determined by DLS (water, 25 °C). PEG, polyethylene glycol, PPO, polypropyleneoxide, RhB, rhodamine B, TEOS, tetraethyl orthosilicate, TMSCl, trimethylchlorosilane.
Figure 2
Figure 2
(A) Cell viability of peptide–NPs as % of control cells. Bars represent the mean ± SD from two independent experiments (n = 2), each performed in triple. (B) Caspase-9 levels after 6 h of incubation with either 1 µM AVPI/cRGD–NPs or AVPI–NPs. Bars indicate the increase in activity of the treated cells compared to the control. Data are reported as mean ± SD from at least three independent experiments. *** p < 0.001.
Figure 3
Figure 3
Fluorescence microscopy of cells after 1 h of treatment with fluorescent peptide–RhB–NPs (red), counterstained with anti α-tubulin antibody (green) to visualize the cytoskeleton. (A) HT29 cells. Photographs were taken at 60× magnification, bar = 20 µm. (B) RhB-positive HT29 cells %, error bars represent SD (n = 15 imaging fields), *** p < 0.001. (C) HT29 cell viability % in the presence of AVPI–NPs; data represent mean ± SD (n = 6). * p < 0.05. (D) HeLa cells. Photographs were taken at 40× magnification. Bar 20 µm. (E) RhB-positive HeLa cells %, error bars represent SD (n = 15 imaging fields), *** p < 0.001. (F) HeLa cells were treated or not with an anti-CD49e antibody or control IgG for 1 h, then incubated whit fluorescent AVPI/cRGD–NPs (red) and counterstained with Hoechst33342 dye (blue) to visualize the nuclei. Photographs were taken at 40× magnification. (G) HeLa cells internalization %, error bars represent SD (n = 15 imaging fields), bar = 20 µm, *** p < 0.001.
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