Aminofullerenes as targeted inhibitors of EGFR: from pancreatic cancer inhibitors to Drosophila m. Toxicology

Abstract

Aim: Pancreatic ductal adenocarcinoma (PDAC) is recognized as one of the most formidable cancers, largely due to its distinct microenvironment characterized predominantly by extensive desmoplastic stroma. In this study, we synthesized three novel water-soluble fullerene-based nanomaterials targeting EGFR protein.

Methods: The direct amination of fullerene carbon atoms, was followed by conjugation with a modified derivative of the EGFR inhibitor-erlotinib, resulting in the formation of novel water-soluble fullerene derivatives.

Results: Further investigation into PAN02 and AsPC-1 cell lines revealed that these fullerene nanomaterials could induce cell cycle arrest in the G0/G1 phase, corroborated by alterations in the expression levels of the p27 and cyclin E1 proteins. Additionally, mechanisms of cell death were identified as autophagy for C₆₀BUT and C₇₀BUT-ERL, and apoptosis for Gd@C₈₂EDA-ERL nanomaterials.

Conclusions: Crucially, the study uncovered the efficacy of synthesized aminofullerenes in inhibiting the EGFR signaling pathway. The further toxicological studies of Gd@C₈₂EDA-ERL fullerene on Drosophila melanogaster, underscored its potential for theranostic applications.

Keywords: EGFR inhibitors; Fullerene; aminofullerene; cancer nanotechnology; drosophila; pancreatic cancer.

Graphical abstract

Figure 1.

Roadmap of the current study: (A) synthesis of fullerene nanomaterials and their in vitro activity with mechanism of action (EGFR signaling inhibition) and in vivo toxicology studies. Created with Biorender^R.

Figure 2.

Spectroscopic characterization of synthesized aminofullerenes: UV-VIS spectra of obtained fullerene nanomaterials (a–c), and FT-IR spectra of synthesized compounds (d–f).

Figure 3.

The XPS C 1s, N 1s, Gd 3d5/2, and Cl 2p lines of C₆₀BUT, C₇₀BUT-ERL, and Gd@C₈₂EDA-ERL fullerene derivatives.

Figure 4.

The SEM (a–c), cryo-EM (b, scale bar 20 nm), and EDS images (d) of synthesized aminofullerenes. The spherical aggregates of C₆₀BUT (a) and C₇₀BUT-ERL (b) as well as fluffy-type aggregates of Gd@C₈₂EDA-ERL (c) were observed.

Figure 5.

The r₁ and r₂ relaxivities of synthesized gadofullerenes.

Figure 6.

The antiproliferative activity of tested nanomaterials (C₆₀BUT, C₇₀BUT-ERL, and Gd@C₈₂EDA-ERL), ligand ERL-COOH and erlotinib against pancreatic cells: PANC-1, PAN02, AsPC-1, and NHDF (a). The impact of tested nanomaterials on the progression of cell cycle (b) and apoptosis induction (c) in PAN02 and AsPC-1 cells. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with the untreated cells (control).

Figure 7.

The level of protein expression after exposure to fullerene nanomaterials: C₆₀BUT, C₇₀BUT-ERL, and Gd@C₈₂EDA-ERL in PAN02 and AsPC-1. Densitometric analyses of these results as well as uncropped and unmodified blots were provided in the Supporting Information.

Figure 8.

Midgut of D. melanogaster. (a-b) FC group (control group). Apical (a) and perinuclear (b) cytoplasm of midgut enterocytes. (c-d) F1W experimental group. Specimens fed with fullerenes for 1 week. Mitochondria (m), microvilli (mv), nucleus (n), cisterns of endoplasmic reticulum (ER), vesicles (v), reserve material (rm), electron-dense granules (black arrows). (a) Scale bar = 1.2 µm. (b) Scale bar = 1.2 µm. (c) Scale bar = 1 µm. (d) Scale bar = 0.8 µm.